MEK Kinase 3 Directly Activates MKK6 and MKK7, Specific
Activators of the p38 and c-Jun NH2-terminal Kinases*
Karl
Deacon and
Jonathan L.
Blank
From the Department of Cell Physiology and Pharmacology, University
of Leicester School of Medicine, P. O. Box 138, Medical Sciences
Building, University Road, Leicester LE1 9HN, United Kingdom
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ABSTRACT |
Mitogen-activated protein kinase
(MAPK)/extracellular signal-regulated kinase
kinase kinase 3 (MEKK3) activates the c-Jun NH2-terminal kinase (JNK) pathway, although no
substrates for MEKK3 have been identified. We have examined the
regulation by MEKK3 of MAPK kinase 7 (MKK7) and MKK6, two novel MAPK
kinases specific for JNK and p38, respectively. Coexpression of MKK7
with MEKK3 in COS-7 cells enhanced MKK7 autophosphorylation and its ability to activate recombinant JNK1 in vitro. MKK6
autophosphorylation and in vitro activation of p38
were
also observed following coexpression of MKK6 with MEKK3. MEKK2, a
closely related homologue of MEKK3, also activated MKK7 and MKK6 in
COS-7 cells. Importantly, immunoprecipitates of either MEKK3 or MEKK2
directly activated recombinant MKK7 and MKK6 in vitro.
These data identify MEKK3 as a MAPK kinase kinase specific for MKK7 and
MKK6 in the JNK and p38 pathways. We have also examined whether MEKK3
or MEKK2 activates p38 in intact cells using MAPK-activated protein
kinase-2 (MAPKAPK2) as an affinity ligand and substrate. Anisomycin,
sorbitol, or the expression of MEKK3 in HEK293 cells enhanced
MAPKAPK2 phosphorylation, whereas MEKK2 was less effective.
Furthermore, MAPKAPK2 phosphorylation induced by MEKK3 or cellular
stress was abolished by the p38 inhibitor SB-203580, suggesting that
MEKK3 is coupled to p38 activation in intact cells.
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INTRODUCTION |
Multiple mitogen-activated protein kinase
(MAPK)1 signaling pathways
have been identified in mammalian cells that are activated in response
to extracellular stimuli and cellular stress (for reviews, see Refs.
1-4). MAPK family members include the extracellular signal-regulated
kinases (ERKs) (1, 2), the c-Jun NH2-terminal kinases
(JNKs) (5, 6), and the p38 kinases (7-12). ERKs are activated by
agonists for tyrosine kinase-encoded receptors and G protein-coupled
receptors that induce mitogenesis, differentiation, or hypertrophy (1,
2), whereas the JNK and p38 subgroups are activated by cellular stress
(e.g. UV and
-irradiation, osmotic stress, heat shock,
protein synthesis inhibitors) and inflammatory cytokines
(e.g. TNF-
and interleukin-1) (3-9, 11, 12). The stress-activated kinases have been implicated in apoptosis (3-15), oncogenic transformation (16-18), and inflammatory responses (19, 20)
in various cell types.
MAPK activation requires dual phosphorylation on Thr and Tyr within the
motif Thr-Xaa-Tyr, where Xaa represents Glu in ERK, Pro in JNK, and Gly
in p38 (2, 3). Specific MAPK kinases have been identified for each MAPK
subgroup that allow for their selective activation (2, 3). Thus MEK1
and MEK2 selectively phosphorylate and activate the ERK subgroup (21,
22), whereas MKK3 and MKK6 selectively activate p38 (8, 9, 11, 12, 22-25). MKK4 (also known as stress-activated protein kinase/ERK kinase
1 or JNK kinase) does not activate the ERK subgroup but activates both
p38 and JNK (8, 22, 25-27). A second JNK kinase, designated MKK7, has
recently been identified as a specific activator of the JNK subgroup
(28-31).
In turn, activation of MAPK kinases involves dual phosphorylation on
Thr and/or Ser residues within a conserved sequence located between
kinase subdomains VII and VIII (22, 32-35). Raf-1 is a MAPK kinase
kinase (MAPKKK) that activates ERK via direct MEK phosphorylation at
these sites (32-34). Raf-1 activation by cell surface receptors
involves Ras-GTP, which binds to the NH2-terminal domain of
Raf-1, causing its translocation and activation at the plasma membrane
(1, 2, 32). Other Raf isoforms include A-Raf and B-Raf, and these also
activate the MEK/ERK cascade (2, 32). Indeed, evidence to date suggests
that Raf kinases represent the primary activators of the MEK/ERK
pathway in vivo.
Additional MAPKKKs exist in mammalian cells that differ structurally
from Raf and preferentially regulate the stress-activated protein
kinase pathways (3, 4). Members of the MEKK family of serine/threonine
kinases were the first MAPKKKs shown to activate JNK in cells (36) and
directly phosphorylate MKK4 in vitro (27, 35, 37-39). Other
MAPKKKs that directly activate MKK4 in the JNK cascade include tumor
progression locus-2 (40); transforming growth factor-
-activated
kinase-1 (TAK-1) (41); the mixed lineage kinases MLK-2 (42), MLK-3
(43), and MAPK upstream kinase (42); MAPK kinase kinase-5 (MAPKKK5)
(44); apoptosis signal-regulating kinase-1 (ASK1) (45); and a human
MEKK homologue termed MAP three kinase-1 (46). Additionally, ASK1 (45),
MAPK three kinase-1 (46), and TAK1 (24) have been demonstrated to
activate p38 and to directly phosphorylate MKK3 and/or MKK6 in
vitro. Thus, there exists a tremendous diversity in the potential
pathways by which MEKKs and related MAPKKKs regulate the
stress-activated protein kinases in a stimulus and cell type-specific manner.
Four mammalian MEKK isoforms have been identified by virtue of their
sequence homology with Ste11 and Byr2 (38, 39, 47), protein kinases
involved in the pheromone mating response pathways in yeast. The
cDNAs for MEKK1 and -4 encode proteins of 161 (47) and 180 kDa
(39), whereas those for MEKK2 and -3 predict proteins of 70 and 71 kDa,
respectively (38). MEKK2 and -3 are closely related in primary
sequence, sharing 77% identity overall and 94% identity between their
carboxyl-terminal catalytic domains (38). MEKK2 and -3 are less closely
related to other MEKKs, showing approximately 50% identity to MEKK1
and -4 through their respective catalytic domains (39). Recent work has
established that MEKK1, -2, and -4 can activate JNK and directly
phosphorylate and activate MKK4 (27, 35, 37-39), whereas MEKK3
strongly induces JNK activity in cells but is inactive with MKK4
in vitro or, indeed, with any other substrate so far
examined (MEK1, MEK2, MKK3) (35, 38). These observations suggest that
MEKK3 may mediate JNK activation via a novel MAPK kinase distinct from
MKK4. In the present study we have examined the ability of MEKK3 to
regulate MKK7 (28-31) and MKK6 (23-25), two recently identified MAPK
kinases specific for the JNK and p38 subgroups. We show that MEKK3 is a
MAPKKK that preferentially activates these novel stress-activated
protein kinase cascades.
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EXPERIMENTAL PROCEDURES |
Plasmids--
Constructs for bacterial expression of MKK3, MKK4,
MKK6, MKK7, JNK1, p38
, ATF2, and c-Jun as translational fusions with
glutathione S-transferase (GST) were kind gifts from Dr.
Roger J. Davis and have been described previously (22, 23, 28). The
bacterial expression vector encoding amino acids 46-400 of MAPKAPK2
and fused to GST (48) was generously provided by Prof. Christopher J. Marshall. The GST fusion proteins were purified by affinity chromatography (49) on glutathione-Sepharose (Amersham Pharmacia Biotech). Purified fusion proteins were resolved by SDS-polyacrylamide gel electrophoresis using 10 or 12% polyacrylamide in the presence of
0.1% SDS (50) and quantified by comparative Coomassie Blue staining
using bovine serum albumin to construct a standard curve. The Flag
epitope tag DYKDDDDK was introduced between codons 1 and 2 of MEKK2 and
MEKK3 by insertional overlapping polymerase chain reaction and cloned
into the polylinker of pCMV5 (51) for mammalian expression as described
(35). Flag-tagged MKK6 and MKK7 were similarly prepared (23, 28) and
expressed from pCDNA3 (Invitrogen).
Cell Culture and Transfection--
COS-7 cells were maintained
in Dulbecco's modified Eagle's medium at 37 °C in a 5%
CO2, 95% air mixture. HEK293 cells were maintained under
similar conditions in minimum essential medium (Life Technologies,
Inc.). Media were supplemented with 10% fetal bovine serum, 50 IU/ml
penicillin, 50 µg/ml streptomycin, 2.5 µg/ml amphotericin B, and 2 mM L-glutamine (Life Technologies, Inc.). Cells
were transfected with plasmid DNA as described previously (35).
Immunoprecipitation--
Protein kinases were isolated by
immunoprecipitation from cell lysates as described previously (35).
Briefly, cells were solubilized with lysis buffer containing 20 mM Tris-HCl, pH 7.4, 1% Triton X-100, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 25 mM
-glycerophosphate, 1 mM Na3VO4,
1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin. To recover epitope-tagged kinases, cleared lysates were
incubated for 1 h at 4 °C with 1 µg of anti-Flag M2
monoclonal antiserum (sc-7787, Santa Cruz Biotechnology). To measure
native p38 kinase activity, lysates were incubated for 1 h at
4 °C with 0.5 µg of a rabbit polyclonal antiserum directed against
the carboxyl-terminal sequence of p38
(sc-535, Santa Cruz
Biotechnology). Immune complexes were incubated further for 1 h at
4 °C with protein A-Sepharose (Amersham Pharmacia Biotech) and
recovered by centrifugation. Immunoprecipitates were washed twice in
200 µl of lysis buffer and twice in 200 µl of kinase buffer,
containing 25 mM HEPES, pH 7.2, 25 mM
-glycerophosphate, 25 mM MgCl2, 2 mM dithiothreitol, and 100 µM
Na3VO4.
Immune Complex Kinase Assays--
Immune complex protein kinase
assays were performed in a final volume of 40 µl of kinase buffer.
Protein kinase reactions were initiated by the addition of the
appropriate recombinant substrate proteins (5 µg each of GST-c-Jun
and GST-ATF2; 1 µg each of GST-JNK, GST-p38
, GST-MKK3, and
GST-MKK4; 0.5 µg each of GST-MKK6 and GST-MKK7) and kinase buffer
containing 250 µM ATP and 5 µCi of
[
-32P]ATP. Reactions were incubated for 20 min at
30 °C and terminated by the addition of an equal volume of Laemmli
sample buffer and boiling. Phosphorylated proteins were resolved by
SDS-polyacrylamide gel electrophoresis through 10% polyacrylamide and
visualized by autoradiography. Data shown in each figure are
representative of 2-4 independent experiments.
p38 Kinase Assay--
p38 activity was assessed using
GST-MAPKAPK2(46-400) as an affinity ligand and substrate (8, 48, 52).
Cleared cell lysates, prepared as for immunoprecipitation, were
incubated with for 60 min at 4 °C with a slurry of
glutathione-Sepharose precoupled to GST-MAPKAPK2 (1 µg of protein).
Beads were collected, washed, and incubated in the presence of
[
-32P]ATP exactly as described for immune complex
kinase assays, except that no further addition of recombinant protein
substrates was required.
Antisera--
Rabbit antiserum B161 was raised against a
purified recombinant GST fusion protein containing amino acids 1-290
of MEKK2 (38). Rabbit polyclonal antisera selective for ERK1 (sc-93)
and JNK1 (sc-474) were obtained from Santa Cruz Biotechnology.
Western Blot Analysis--
Immunoblot analysis of cleared cell
lysates was performed as described previously (35). Analysis of MAPK
proteins isolated from HEK293 cells by affinity binding to GST-MAPKAPK2
was performed following incubation and washing of the
glutathione-Sepharose matrix as described under "p38 Kinase Assay."
Immunoreactivity was visualized using enhanced chemiluminescence
reagents (Amersham Pharmacia Biotech).
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RESULTS |
Activation of MKK6 and MKK7 by MEKK3 and MEKK2 in COS-7
Cells--
Flag epitope-tagged MKK6 and MKK7 were separately expressed
in COS-7 cells either alone or in combination with MEKK3 or MEKK2. The
MKKs were then isolated by immunoprecipitation using a monoclonal antiserum to the Flag epitope and their protein kinase activities assessed in vitro. Fig.
1A shows that MKK6
autophosphorylation was low relative to that of MKK7. However, the
autocatalytic activities of both MKK6 and MKK7 were enhanced by their
coexpression with either MEKK3 or MEKK2. MKK6 and MKK7 activity was
directly assessed in the immune complex kinase assay using purified
recombinant p38
or JNK1 as substrates, respectively. These data
confirmed that the activities of MKK6 and MKK7 were enhanced by their
coexpression with either MEKK3 or MEKK2, although p38
appeared to
act as a better substrate for MKK6 than did JNK1 for MKK7 (Fig.
1B). Western blot analysis using the anti-Flag antibody
showed that expression of MKK6 or MKK7 was essentially unaffected by
MEKK coexpression (Fig. 1C). An antiserum (B161), which at
low dilution recognizes both MEKK2 and MEKK3, confirmed expression of
these kinases in the appropriate cell lysates (Fig. 1C).

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Fig. 1.
Enhanced autophosphorylation and activation
of MKK6 and MKK7 by MEKK3 and MEKK2. COS-7 cells were transfected
with empty vector (pCMV5, lanes 1 and 5), Flag-MKK6
(lanes 2-4), or Flag-MKK7 (lanes 6-8) in the
presence of empty vector (pCMV5, lanes 2 and 6),
MEKK3 (lanes 3 and 7), or MEKK2 (lanes
4 and 8). Flag-MKK6 and Flag-MKK7 were isolated from
lysates by immunoprecipitation, and their protein kinase activities
were assessed in vitro as described under "Experimental
Procedures." A, effect of MEKK3 or MEKK2 on Flag-MKK6 and
Flag-MKK7 autophosphorylation. B, effect of MEKK3 or MEKK2
on phosphorylation of GST-p38 and GST-JNK1 by Flag-MKK6 and
Flag-MKK7, respectively. C, Western blot analysis of
Flag-MKK and MEKK expression using anti-Flag M2 and B161
antisera.
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To determine whether MKK-catalyzed phosphorylation of recombinant JNK1
and p38
resulted in activation of these MAPKs, immune complex kinase
assays of Flag-tagged MKK6 and MKK7 were performed in which the
transcription factors ATF2 or c-Jun were provided as in
vitro substrates for p38
or JNK1, respectively. Fig.
2A shows that ATF2
phosphorylation by p38
was stimulated by immunoprecipitates of MKK6
obtained from cells coexpressing either MEKK3 or MEKK2. Similar data
were obtained in parallel experiments in which JNK1 and c-Jun were used
as the sequential substrates of MKK7 (Fig. 2B). Omission of
either p38
(Fig. 2A) or JNK1 (Fig. 2B) from the in vitro reactions showed that the activated MKKs did
not directly phosphorylate ATF2 or c-Jun, thereby confirming the
specificity of the assays. Thus, MEKK2 and MEKK3 are both capable of
activating MKK6 and MKK7 in COS-7 cells.

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Fig. 2.
MEKK3 and MEKK2 can activate
p38 and JNK1 via MKK6 and MKK7.
A and B, COS-7 cells were transfected with empty
vector (pCMV5, lanes 1, 2, 9, and 10), Flag-MKK6
(lanes 3-8), or Flag-MKK7 (lanes 11-16) in the
presence of empty vector (pCMV5, lanes 3, 4, 11, and
12), MEKK3 (lanes 5, 6, 13, and 14),
or MEKK2 (lanes 7, 8, 15, and 16). Flag-MKK6 and
Flag-MKK7 were isolated by immunoprecipitation and assayed using either
GST-p38 (A, lanes 1, 3, 5, and 7)
or GST-JNK1 (B, lanes 9, 11, 13, and
15) as in vitro substrates. Activation of
GST-p38 and GST-JNK1 was assessed using GST-ATF2 (A) and
GST-c-Jun (B) as substrates.
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MEKK3 Directly Activates MKK6 and MKK7 in Vitro--
Previous
studies have failed to identify any direct substrates of MEKK (35, 38),
although cotransfection experiments have shown that MKK3 and MKK4 can
be activated by MEKK3 in COS-7 cells (35). Because MEKK3 also activates
MKK6 and MKK7 in intact cells (Fig. 2), we determined whether MEKK3 was
capable of directly activating MKK6 or MKK7 in vitro. Flag
epitope-tagged MEKK3 was expressed in COS-7 cells and isolated by
immunoprecipitation. MEKK activity was then assessed in the immune
complex kinase assay using recombinant MKK3, MKK4, MKK6, or MKK7 as
substrates. To fully reconstitute each activation pathway, recombinant
JNK1 and c-Jun were provided with MKK4 or MKK7, whereas recombinant
p38
and ATF2 were provided with MKK3 or MKK6. Consistent with
previous studies (35), immunoprecipitated MEKK3 failed to activate MKK3 or MKK4 in vitro (Fig.
3A). By contrast,
immunoprecipitates of MEKK3 directly activated MKK6 and MKK7, resulting
in a large increase in phosphorylation of the relevant transcription
factor by p38
and JNK1, respectively (Fig. 3A).
Immunoblot analysis showed equivalent expression of the Flag-MEKK2
protein used for the reconstitution assays (Fig. 3B). Thus,
MEKK3 is a MAPK kinase kinase that shows selectivity in its activation
of MKK6 and MKK7 over MKK3 or MKK4 in vitro.

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Fig. 3.
MEKK3 directly activates MKK6 and MKK7
in vitro but not MKK3 or MKK4. COS-7 cells were
transfected with either empty vector (pCMV5, lanes 1, 3, 5, and 7) or Flag-MEKK3 (lanes 2, 4, 6, and
8). A, Flag-MEKK3 was isolated by
immunoprecipitation and assayed using GST-MKK3 (lane 2),
GST-MKK4 (lane 4), GST-MKK6 (lane 6), or GST-MKK7
(lane 8) as in vitro substrates. Activation of
GST-MKK3 and GST-MKK6 was assessed using GST-p38 and GST-ATF2 as
sequential substrates (lanes 1, 2, 5, and 6),
whereas GST-MKK4 and GST-MKK7 activity was assessed using GST-JNK1 and
GST-c-Jun as substrates (lanes 3, 4, 7, and 8).
B, Flag-MEKK3 expression was detected by immunoblotting with
anti-Flag M2 antibody.
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Immunoprecipitates of Flag-tagged MEKK2 from COS-7 cells strongly
autophosphorylated and also directly activated MKK6 and MKK7 when the
kinase cascades were reconstituted using the relevant MAPKs and
transcription factors in vitro (Fig.
4A). Omission of MKK7 and/or
JNK1 prevented c-Jun phosphorylation (Fig. 4A), indicating that MEKK2 did not activate JNK1 or phosphorylate c-Jun directly. Similarly, ATF2 phosphorylation by p38
was only observed when MKK6
was reconstituted with MEKK2 (Fig. 4A). Western blot
analysis confirmed equivalent expression of the Flag-MEKK2 protein used for reconstitution (Fig. 4B). Thus, these data indicate that
MEKK2 is also capable of activating MKK6 and MKK7 in vitro
and in intact cells.

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Fig. 4.
MEKK2 directly activates MKK6 and MKK7
in vitro. COS-7 cells were transfected with
either empty vector (pCMV5, lanes 1 and 6) or
Flag-MEKK2 (lanes 2-5 and 7-10). A,
Flag-MEKK2 was isolated by immunoprecipitation and assayed using
GST-MKK7 (lane 2 and 5) or GST-MKK6 (lanes
7 and 10) as in vitro substrates. Activation
of GST-MKK7 was assessed using GST-JNK1 and GST-c-Jun as sequential
substrates (lane 2), whereas GST-MKK6 activity was assessed
using GST-p38 and GST-ATF2 as substrates (lane 7).
Control assays were performed in which the MKK (lanes 3, 4, 8, and 9) and/or the MAPK (lanes 4, 5, 9, and 10) was omitted from the in vitro reaction.
B, Flag-MEKK2 expression was detected by immunoblotting with
anti-Flag M2 antibody.
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Activated MKK6 and MKK7 Are Specific for p38 and JNK,
Respectively--
To examine the specificity of MKK6 and MKK7 for
p38
and JNK1, Flag-MEKK3 was isolated from COS-7 cells by
immunoprecipitation and used to activate purified MKK6 and MKK7
in vitro. Each MKK was provided with either recombinant JNK1
or p38
together with their substrates c-Jun or ATF2, respectively.
These in vitro kinase assays indicated that MKK6 activated
p38
but not JNK1, whereas MKK7 activated JNK1 but not p38
(Fig.
5). These data confirm that MKK6 and MKK7
are specific for p38
and JNK1, respectively, and that this
specificity is maintained following MEKK activation.

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Fig. 5.
MKK7 and MKK6 are specific for JNK1 and
p38 following activation by MEKK3. COS-7
cells were transfected with either empty vector (pCMV5, lanes 1, 3, 5, and 7) or Flag-MEKK3 (lanes 2, 4, 6, and 8). Flag-MEKK3 was isolated by immunoprecipitation and
combined with either GST-MKK7 (lanes 2 and 4) or
GST-MKK6 (lanes 6 and 8) in vitro. The
ability of activated GST-MKK7 and GST-MKK6 to activate GST-JNK1 and
GST-p38 was determined using GST-c-Jun and GST-ATF2 as substrates,
as indicated.
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MEKK3 Is a Potential Activator of p38 in Intact Cells--
MEKK2
and MEKK3 are established activators of the JNK pathway in
vitro and in intact cells (35, 38). However, whereas p38
activation by MEKK2 and MEKK3 can be reconstituted using cognate MKKs
in vitro (Figs. 1-5 and Ref. 35), previous studies have
failed to demonstrate significant activation of p38
by its coexpression with either MEKK2 or MEKK3 in HEK293 cells (35). As
MAPKAPK2 can serve as a substrate for the
,
, and
isoforms of
p38 (8, 10, 48), we have attempted to determine whether MEKK2 or MEKK3
is capable of a stimulating a native p38 kinase activity in HEK293
cells using MAPKAPK2 as an affinity ligand to isolate p38 kinases and
serve as their substrate (8, 10, 48, 52). Fig.
6A shows that MAPKAPK2
phosphorylation was enhanced following treatment of HEK293 cells with
sorbitol or anisomycin, two stress activators of p38
,
,
, and
isozymes (7-12). Furthermore SB-203580, a specific inhibitor of
the p38 subgroup of MAPKs (8, 9, 19, 52), blocked this phosphorylation,
thereby confirming that this assay detected p38 kinase activity.
Additionally, expression of Flag-tagged MEKK2 or MEKK3 enhanced
MAPKAPK2 phosphorylation and this could also be inhibited by SB-203580.
Western blot analysis using anti-Flag monoclonal antiserum confirmed
expression of MEKK3 and MEKK2 in the appropriate cell lysates (Fig.
6A). Immunoblot analysis also indicated that the use of
GST-MAPKAPK2 as an affinity ligand allowed selective enrichment of
p38
from HEK293 cell lysates, although some ERK binding could also
be detected upon prolonged exposure of immunoblots (Fig.
6A). Thus, MEKK3 and MEKK2 may activate a native p38 kinase
in HEK293 cells. Of note, MEKK3 reproducibly induced MAPKAPK2
phosphorylation, whereas MEKK2 was less effective.

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Fig. 6.
MEKK3 is a potential activator of p38 in
HEK293 cells. HEK293 cells were either untransfected (lanes
4-7) or transfected with empty vector (pCMV5, lane 1),
Flag-MEKK3 (lane 2), or Flag-MEKK2 (lane 3).
Untransfected cells were either treated for 15 min with sorbitol (300 mM, lane 4) and anisomycin (10 ng/ml, lane
6) or with their carriers phosphate-buffered saline (PBS)
(lane 5) and Me2SO (DMSO) (lane
7). A, native p38 was isolated by affinity purification
using GST-MAPKAPK2 and assayed using this fusion protein as substrate.
Where indicated, SB-203580 (10 µM) was included in the
in vitro reaction. Flag-MEKK3 and Flag-MEKK2 expression was
detected by immunoblotting with anti-Flag M2 antibody. As shown in the
lower portion of A, immunoblot analysis using anti-p38 ,
anti-JNK1, and anti-ERK1 antisera was performed on total cell lysates
(approximately 10 µg of protein, lanes 3), on washed
GST-MAPKAPK2/glutathione-Sepharose beads that had been preincubated
with HEK293 cell lysate (approximately 100 µg of protein, lanes
2), or with lysis buffer as control (lanes 1). The
relative position of the 45-kDa ovalbumin marker is shown.
B, native p38 was isolated by immunoprecipitation and
assayed using GST-ATF2 as substrate.
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To extend these observations, an antiserum raised to p38
was used to
precipitate native p38 from cells that had been treated with sorbitol
or anisomycin or transfected with Flag-tagged MEKK3 or MEKK2. p38
activity was measured in vitro using ATF2 as substrate. Although recovery of p38 kinase activity was low, detectable p38 activation could be observed by anisomycin, sorbitol, and MEKK3 (Fig.
6B). However, the potency of the effect did not correlate well with MAPKAPK2 phosphorylation, suggesting that MAPKAPK2 may precipitate additional isozymes of p38 that are not immunoprecipitated by the p38
-selective antiserum.
 |
DISCUSSION |
Recent molecular cloning studies have revealed the existence of
numerous MAPKKKs in mammalian cells (1-4, 32, 38-47). It seems likely
that this diversity provides multiple mechanisms for activation of MAPK
subgroups in a stimulus- and cell type-specific manner. In most cases,
MAPK regulation by these MAPKKKs correlates with their ability to
phosphorylate and activate MAPKKs selective for each subgroup of MAPK.
Thus Raf-1 is a MAPKKK that directly phosphorylates and activates MEK1
and MEK2 in the ERK pathway (32-34), whereas the JNK activator, MKK4,
does not serve as a Raf-1 substrate (27). Conversely, MEKK isozymes can
function as MAPKKKs in the ERK and JNK signaling pathways (27, 33,
35-39). Several studies indicate that MEKK1 may activate MKK4 and/or
JNK in preference to MEK and/or ERK (36, 37, 47), whereas MEKK2 and
MEKK3 strongly activate both the JNK and ERK pathways (38). MEKK4 shows
the greatest selectively for the MKK4/JNK cascade and does not appear
to activate MEK or ERK (39). Other MAPKKKs that activate JNK have also
been demonstrated to directly phosphorylate and activate MKK4 in
vitro, including tumor progression locus-2 (40), TAK-1 (41), MLK-2
(42), MLK-3 (43), MAPK upstream kinase (42), MAPKKK5 (44), ASK1 (45),
and the human homologue of MEKK4 termed MAPK three kinase-1 (46).
However, JNK activation by MEKK3 appears atypical in that it does not
appear to involve MKK4 (35, 38). Indeed, despite its close homology to
MEKK2, a direct activator of MEK1, MEK2, and MKK4, previous studies
have failed to identify any MAPKK as a substrate for MEKK3 (35,
38).
Recent work has identified MKK7 as a specific activator of JNK
(28-31). This selectivity differs from that of MKK4, which is capable
of activating the JNK and p38 subgroups (8, 22, 25-27). In the present
study, we have addressed whether MEKK3 and its close structural
homologue, MEKK2, are capable of regulating MKK7. Our data show that
MEKK3 and MEKK2 are both capable of activating MKK7 in intact cells and
in vitro. Thus, MEKK3 represents a MAPKKK that selectively
activates MKK7 in the JNK pathway, whereas MEKK2 is also capable of
activating MKK4. Very recently, MEKK1 and MLK2 have also been shown to
directly activate MKK7 (53). Interestingly, whereas MEKK1 activates
MKK7 and MKK4 to similar extents, MLK2 appears to be a more effective
activator of MKK7 than MKK4 (53). Thus, the selectivity of MEKK3
appears to be similar to that of MLK2, except that MEKK3 shows an
absolute specificity for MKK7 over MKK4. As has been postulated for
MLK2 (53), the specificity shown by MEKK3 may enable selective
activation of MKK7 by extracellular stimuli and cellular stress. The
observation of MKK7 activation by TNF-
in the absence of detectable
MKK4 stimulation (31) suggests that MEKK3 may be involved in the
preferential activation of MKK7 initiated by such a stimulus. Of note
the JNK-interacting protein, JIP, is capable of binding JNK, MKK7, and
MEKK3 but not MKK4 (54). The proposed role of JIP as a molecular
scaffold (54) is consistent with the substrate selectivity of MEKK3 for MKK7 and may facilitate JNK signaling via this cascade.
Certain MAPKKKs have been identified as components of p38 signaling
pathways. Those shown to activate p38 and directly phosphorylate and
activate MKK3 and/or MKK6 include TAK1 (24) and ASK1 (45). Most studies
indicate that MEKK types 1-4 do not activate p38 (27, 35, 38, 39),
although MEKK1 (47, 55) and MAPK three kinase-1 (human MEKK4) (46) may
cause modest activation of p38 cascades. Previous studies from this
laboratory indicate that neither MEKK2 nor MEKK3 is capable of directly
activating MKK3 in vitro (35), consistent with their lack of
effect on p38 kinase activity in intact cells. It is surprising,
therefore, that MEKK3 is capable of activating MKK3 when their
corresponding cDNAs are transiently coexpressed in COS-7 or HEK293
cells. Activation of MKK3 requires its phosphorylation on Ser-189 and
Thr-193 and is specific for MEKK3 in that MEKK2 is without effect in
parallel experiments (35). However, the failure of MEKK3 to activate MKK3 in vitro suggests that the effects of MEKK3 on MKK3
activity in cells may be indirect.
To further define the function of MEKK3 and MEKK2 in p38 signaling, we
have examined the regulation of MKK6 by these MEK kinases. We show that
MEKK3 and MEKK2 can activate MKK6 in intact cells and in
vitro. Thus, MEKK3 and MEKK2 are candidate MAPKKKs for the
MKK6/p38 signaling pathway. Based on the apparent specificity of MEKK3
and MEKK2 for MKK6 as a target substrate, we have re-examined the
potential for p38 regulation by both MEKKs. Four principle isoforms of
p38 have been identified and are designated
(7),
(8, 9),
(10), and
(11, 12). Interestingly, recent evidence suggests that
individual p38 subtypes may be differentially activated by MKK3 and
MKK6 (8, 9, 11). Other work has demonstrated that p38
can
phosphorylate MAPKAPK2 at Thr-222, Ser-272, and Thr-344 in
vitro, resulting in activation of the kinase (48). In addition,
activation of MAPKAPK2 in cells can be blocked by SB-203580 (48), a
pyridyl imidazole compound that inhibits p38 kinase activity in cells
(19, 52) and directly inhibits p38
,
1, and
2 isoforms
in vitro (8-10). MAPKAPK3 is also activated by p38 in
vitro and in vivo and can associate directly with
recombinant and endogenous forms of p38 (56). Thus, we have examined
the ability of MEKK3 and MEKK2 to activate native p38 in HEK293 cells
using MAPKAPK2 as an affinity ligand and a substrate. Our data indicate
that GST-MAPKAPK2 can be used to selectively isolate endogenous p38
from cells, as demonstrated for GST-MAPKAPK3 previously (56).
Furthermore MAPKAPK2 served as an in vitro substrate for
associated kinase(s) that were activated by sorbitol, anisomycin,
MEKK3, and MEKK2 to a lesser extent. Importantly, this MAPKAPK2
phosphorylation was blocked by the p38 inhibitor SB-203580. Thus, these
studies suggest that MEKK3 and MEKK2 are coupled to MKK6 and p38
activation in vivo. As SB-203850 does not inhibit ERK, JNK
(19), p38
(9, 10), or p38
(12), the endogenous p38 activity
induced by MEKK3 and MEKK2 may include the p38
and/or p38
subtypes. Although immunoprecipitates of native p38
could be
activated by MEKK3 and MEKK2, these effects were modest and have not
been apparent by coexpression of either MEKK with recombinant p38
(35). Clearly, identification of the p38 isoform(s) regulated by MEKKs
merits further investigation.
Although the intracellular mechanisms involved in MEKK regulation
remain largely unknown, the noncatalytic NH2-terminal
domains of MEKK1-4 are highly divergent, suggesting unique regulatory roles for each. For example, this domain in MEKK1 allows for its association with the particulate fraction and contains two proline-rich regions, a cysteine-rich region, and two potential pleckstrin homology
domains (47). The NH2-terminal region of MEKK1 is
inhibitory as caspase-dependent cleavage at the DTVD
sequence located between amino acids 870 and 875 in MEKK1 activates the
enzyme (57). This mechanism may account for MEKK1-mediated JNK
activation and anoikis when Madin-Darby canine kidney cells lose
contact with the extracellular matrix (57). The
NH2-terminal region of MEKK4 has low homology to that of
MEKK1 but shares a proline-rich region and potential pleckstrin
homology domain (39). In addition, MEKK1 and MEKK4 bind Rac1 and Cdc42
(39, 58), and MEKK4 contains a CRIB (Cdc42 or
Rac interactive binding)-like
motif, which may confer it with this ability (39). By contrast, MEKK2
and -3 contain shorter noncatalytic moieties that lack the putative
regulatory domains present in either MEKK1 or MEKK4 (38). Recently,
MEKK1, MEKK2, and MEKK3 have been reported to bind 14-3-3 proteins
(59), although the functional significance of these interactions are unknown.
Certain MAPKKKs have been implicated in JNK and p38 pathways regulated
by cell surface receptors and cellular stresses. For example, MEKK1 can
be activated by epidermal growth factor (60), cross-linking of high
affinity IgE receptors (61), and TNF-
(62) in various cell types.
Similarly, TAK1 can be activated by transforming growth factor-
(41), UV, sorbitol, ceramide, TNF-
, interleukin-1, and Fas receptor
ligation (63). ASK-1, which is similar biochemically to TAK1, also
appears to be activated by TNF-
(45). Although one report suggests
that MEKK2 can be activated by epidermal growth factor (59), the
mechanisms involved in MEKK2 and MEKK3 regulation remain to be defined.
The identification of MKK6 and MKK7 as the preferential substrates of
MEKK3 will now permit studies to address the involvement of both MEKK3
and MEKK2 in JNK and p38 signaling pathways regulated by extracellular stimuli and cellular stress.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Roger J. Davis for his
generous gifts of MKK3, MKK4, MKK6, MKK7, p38, JNK1, ATF2, and c-Jun
constructs. We also thank Prof. Christopher J. Marshall for the
GST-MAPKAPK2 expression plasmid and Dr. Robin Plevin for helpful
discussions. We are grateful to SmithKline Beecham Pharmaceuticals for
providing the SB-203580 compound.
 |
FOOTNOTES |
*
This work was supported by Grant 99507498MA from the Medical
Research Council and Grant 055813 from the Wellcome Trust.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44 116 252 5198;
Fax: 44 116 252 5045; E-mail: jb48{at}le.ac.uk.
 |
ABBREVIATIONS |
The abbreviations used are:
MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated
protein kinase;
JNK, c-Jun NH2-terminal kinase;
MEK, MAPK/ERK kinase;
MKK, MAPK kinase;
MAPKKK, MAPK kinase kinase;
MEKK, MAPK/ERK kinase kinase;
TAK, transforming growth factor-
-activated
kinase;
MLK, mixed lineage kinase;
ASK, apoptosis signal-regulating
kinase;
MAPKAPK, MAPK-activated protein kinase;
ATF, activating
transcription factor;
GST, glutathione S-transferase;
TNF, tumor necrosis factor.
 |
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