(Received for publication, January 14, 1997, and in revised form, March 24, 1997)
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
We previously reported the isolation of cDNAs encoding two mammalian mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) kinase kinases, designated MEKK2 and MEKK3 (Blank, J.L., Gerwins, P., Elliott, E.M., Sather, S. and Johnson, G.L. (1996) J. Biol. Chem. 271, 5361-5368). In the present study, cotransfection experiments were used to examine the regulation by MEKK2 and MEKK3 of the dual specificity MAP kinase kinases, MKK3 and MKK4. MKK3 specifically phosphorylates and activates p38, whereas MKK4 phosphorylates and activates both p38 and JNK. Coexpression of MEKK2 or MEKK3 with MKK4 in COS-7 cells resulted in activation of MKK4, as assessed by enhanced autophosphorylation and by its ability to phosphorylate and activate recombinant JNK1 or p38 in vitro. MKK3 autophosphorylation and activation of p38 was also observed following coexpression of MKK3 with MEKK3, but not with MEKK2. Consistent with these observations, immunoprecipitated MEKK2 directly activated recombinant MKK4 in vitro but failed to activate MKK3. The sites of activating phosphorylation in MKK3 and MKK4 were identified within kinase subdomains VII and VIII. Replacement of Ser189 or Thr193 in MKK3 with Ala abolished autophosphorylation and activation of MKK3 by MEKK3. Analogous mutations in MKK4 indicated that Ser221 and, to a lesser extent, Thr225 were necessary for MKK4 activation by MEKK2 and MEKK3.
These data indicate that MKK3 is preferentially activated by MEKK3, whereas MKK4 is activated both by MEKK2 and MEKK3. Consistent with these observations, MEKK2 and MEKK3 also activated JNK1 in vivo. However, MEKK3 failed to activate p38 when coexpressed in either the absence or presence of MKK3, indicating that MEKK3 is not coupled to p38 activation in vivo. These observations suggest that regulation of p38 and JNK1 pathways by MEKK3 may involve distinct mechanisms to prevent p38 activation but to allow JNK1 activation.
Mammalian cells contain multiple mitogen-activated protein kinase (MAPK)1 signaling pathways that are activated by a diverse array of extracellular stimuli and regulate a variety of cellular processes (for reviews, see Refs. 1-5). MAPKs consist of a family of serine/threonine kinases that includes the extracellular regulated kinases (ERKs) (1, 4), the c-Jun NH2-terminal kinases (JNKs, or stress-activated protein kinases) (5-9), and p38 (5, 10, 11). ERKs are activated by agonists for tyrosine kinase-encoded receptors (e.g. epidermal growth factor and platelet-derived growth factor) and G protein-coupled receptors that induce mitogenesis or cellular differentiation (2-4). ERKs mediate the effects of these agonists by phosphorylating and regulating the activity of a number of proteins, including cytoplasmic enzymes (e.g. phospholipase A2 and p90rsk) and nuclear transcription factors (e.g. Elk-1) (1, 2).
JNKs phosphorylate the NH2-terminal activation domain of
c-Jun and ATF2, increasing their transcriptional activity (5, 6, 9,
12). JNKs are activated preferentially by cellular stress (UV light,
-irradiation, osmotic stress, heat shock, protein synthesis
inhibitors) (5-7, 13-16) and inflammatory cytokines (tumor necrosis
factor-
and interleukin-1) (7-9) but also by G protein-coupled
receptor agonists (17-19), growth factors, and cytoplasmic oncogenes
(v-Src and v-Ras) (6, 20). JNK is implicated in the induction of
apoptotic cell death in a number of cell types (21-23), although
available evidence also suggests that JNKs may mediate the mitogenic
effects of Rac and Cdc42 in Swiss 3T3 cells (24), and v-Src- and
12-induced transformation in NIH3T3 cells (20, 25, 26).
p38 is also activated by cellular stress and inflammatory cytokines and
by lipopolysaccharide (10, 27, 28). Although less is known about p38
function, substrates include MAPK-activated protein kinase-2 and ATF2
(11, 27, 28), and a role for p38 in mediating cytokine production (29)
and apoptosis (21) has been proposed.
MAPKs are activated by dual phosphorylation on Thr and Tyr within the motif Thr-Xaa-Tyr, where Xaa corresponds to Glu in ERK, Pro in JNK, and Gly in p38 (4, 5). The dual specificity kinases involved are mostly specific for each MAPK subgroup, allowing for their independent regulation (4, 5). Thus, MEK1 and MEK2 selectively phosphorylate and activate the ERK subgroup (4, 30-32), whereas MKK3 and MKK6 selectively phosphorylate and activate p38 (11, 32-35). MKK4 (also known as SEK1 or JNKK) does not activate the ERK subgroup but activates both p38 and JNK (32, 34, 36, 37).
Activation of MEK1 involves phosphorylation on serines 218 and 222 within a conserved regulatory region between kinase subdomains VII and VIII (38-41). Raf-1 directly phosphorylates these sites and is probably the primary MEK activator in vivo. In most cases, Raf-1 activation by receptor tyrosine kinases and G protein-coupled receptors involves Ras, which interacts directly with Raf-1, causing translocation and activation of Raf-1 at the plasma membrane (2-4, 38). A-Raf and B-Raf have also been shown to activate the ERK pathway (42), although their pattern of expression is more restricted than that of Raf-1 (43) and their mechanisms of activation are less well understood. However, B-Raf appears to be the major MEK activator in brain (44, 45) and nerve growth factor-treated PC12 cells (46), and A-Raf has been shown to activate MEK1 in cardiac myocytes following endothelin-1 stimulation (47).
Whereas Raf-1 shows no MKK4-stimulating activity (37), members of the mammalian MEKK family of serine/threonine kinases have been demonstrated to activate MKK4 (37, 48, 49). The cDNAs corresponding to three MEKK isoforms were isolated by virtue of their sequence homology with Ste11 and Byr2 (49, 50), protein kinases involved in the pheromone mating response pathway in Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The cDNA corresponding to MEKK1 encodes a protein of 161 kDa (51), whereas those for MEKK2 and MEKK3 encode proteins of 70 and 71 kDa, respectively (49). MEKK2 and MEKK3 are also more closely related in amino acid sequence, being 94% identical to each other and ~50% identical to MEKK1 through their respective catalytic domains (49). When transiently overexpressed, MEKKs induce constitutive activation of JNK and ERK pathways (37, 41, 49-53), but not p38 (37, 49); both immunoprecipitated MEKK1 and MEKK2 can phosphorylate recombinant MKK4 and MEK1 in vitro (37, 48-51), whereas MEKK3 is unable to phosphorylate either substrate (49). In NIH3T3 cells, expression of inducible MEKK1 catalytic domain results in maximal JNK activation in the absence of detectable ERK stimulation (48), suggesting that MEKK1 may preferentially regulate the JNK pathway. Current evidence suggests that MEKK2 and MEKK3 show little or no preference for activating either JNK or ERK pathways (49).
Although the mechanisms of MEKK activation are unknown, MEKK activities
have been implicated in MAPK pathways regulated by a number of
extracellular stimuli in several cell types. For example, an endogenous
MEKK1 is activated in epidermal growth factor-stimulated PC12 cells via
a pathway involving Ras (54). MEKK1 is also activated in mast cells
following aggregation of high affinity IgE receptors (55) and in tumor
necrosis factor--stimulated macrophages (56). Using a
kinase-deficient MEKK1 mutant, evidence for MEKK1 involvement in tumor
necrosis factor-
-induced NF-
B activation in NIH3T3 cells has also
been reported (57). Another serine/threonine kinase shown to activate
MKK4 in vitro is the MEKK-related TAK-1 enzyme, which is
activated in murine osteoblast cells following transforming growth
factor-
stimulation (58). TAK-1 has also recently been shown to
activate MKK3 and MKK6 in vitro and in vivo (35).
A fifth MEKK homologue, designated Tpl-2, has also recently been shown
to activate the ERK and JNK pathways in vivo and to directly
phosphorylate MEK1 and MKK4 in vitro (59).
The JNK activator MKK4 can also activate p38 in vivo and directly phosphorylate and activate p38 in vitro (32, 34, 37). It is surprising, therefore, that MEKK1 and MEKK2, which directly phosphorylate and activate MKK4, fail to activate p38 in vivo (37, 49). However, it remains possible that MKK4 is not a physiologically relevant p38 activator. The recent identification of MKK3 and MKK6 as specific activators of p38 (11, 32-35) allows for a more direct assessment of the regulatory components of this pathway. To date, only TAK-1 has been identified as a MEK kinase activator of MKK3 or MKK6 (35), although the Rho-type G proteins Rac1 and Cdc42 (19, 20, 24, 26, 60-63), the Ste20-related p21-activated protein kinases (20, 61, 62, 64) and germinal center kinase (65), and the mixed linkage kinases (66, 67) are also implicated as upstream regulators of the kinase cascades leading to p38 and JNK activation.
Using transient transfection experiments, we now show that MEKK3 activates MKK3 in vivo and identify Ser189 and Thr193 in MKK3 as essential activating phosphorylation sites. Furthermore, MKK3 is not significantly activated by MEKK2 in vivo or in vitro, indicating that MKK3 is differentially regulated by MEKK2 and MEKK3. We also show that MEKK2 and MEKK3 can activate the MKK4/JNK pathway in vivo and that Ser221 and, to a lesser extent, Thr225 in MKK4 are necessary for activation. However, MEKK3 fails to activate p38 when coexpressed in either the absence or presence of MKK3, suggesting that regulation of p38 and JNK involves distinct mechanisms to oppose sustained p38 stimulation in the presence of MEKK3.
Constructs for bacterial expression of MKK3,
MKK4, JNK1, p38, ATF2, and c-Jun as translational fusions with
glutathione S-transferase (GST) were a kind gift from Dr. R. J. Davis and have been described previously (6, 12, 32). GST-MEKK3 was
prepared with pGEX-3T (Pharmacia Biotech Inc.) and a polymerase chain
reaction fragment encoding amino acids 1-301 of MEKK3. The GST fusion
proteins were purified by affinity chromatography (70) on
glutathione-Sepharose (Pharmacia). Purified fusion proteins were
resolved by SDS-polyacrylamide gel electrophoresis using 10 or 12%
polyacrylamide in the presence of 0.1% SDS (71) 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, MEKK3, MKK3, MKK4, and p38
by insertional overlapping polymerase chain reaction (72). The cDNA
for each epitope-tagged kinase was subcloned into the polylinker of
pCMV5 (73) for mammalian expression. The sequence of each of these
Flag-tagged constructs was confirmed by DNA sequencing. JNK1 cDNA
was transiently expressed as a hemagglutinin (HA) epitope-tagged
protein from pSR (52) and was kindly provided by Dr. G. L. Johnson.
The cDNAs containing the entire coding region of Flag epitope-tagged MKK3 and MKK4 were amplified by polymerase chain reaction and subcloned into pALTER (Promega). Site-directed mutagenesis of MKK3 and MKK4 was performed using the Altered Sites II mutagenesis system (Promega) according to the manufacturer's instructions and confirmed by DNA sequencing. Flag-MKK3 mutants containing single and double point substitutions of Ser189 and Thr193 with alanine (S189A, T193A, S189A/T193A) were subcloned into pCMV5 for mammalian expression. Corresponding single and double point mutants of Flag-MKK4 (S221A, T225A, and S221A/T225A) were similarly expressed from pCMV5.
Cell Culture and TransfectionCOS-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.). COS-7 cells were transfected with 2 µg of each plasmid DNA/plate using the DEAE-dextran/cholorquine method (68). HEK293 cells were transfected with 5 µg of each plasmid DNA/plate by calcium phosphate precipitation (69). Where necessary, the total amount of DNA used for separate transfections was made equivalent with empty pCMV5 vector. 48 h after transfection, COS-7 and HEK293 cells were made quiescent for 16-18 h in the appropriate serum-free medium containing 0.1% bovine serum albumin.
ImmunoprecipitationEpitope-tagged protein kinases were
isolated by immunoprecipitation from cell lysates essentially as
described previously (32). 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. Insoluble material was removed from
cell lyates by centrifugation at 14,000 × g for 10 min
at 4 °C. Lysates were incubated for 1 h at 4 °C with 1 µg
of the relevant antiserum (either anti-Flag M2 (IBI-Kodak) or anti-HA 12CA5 (Boehringer Mannheim)). Immune complexes were incubated further
for 1 h at 4 °C with protein A-Sepharose (Pharmacia) 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 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) 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. Samples were boiled for 5 min, and proteins were
resolved by SDS-polyacrylamide gel electrophoresis through 10%
polyacrylamide. Gels were dried under vacuum, and phosphorylated
proteins were visualized by autoradiography. Data shown in each figure
are representative of 2-5 independent experiments.
Rabbit antiserum B128 was raised against a synthetic peptide corresponding to amino acids 602-619 in the COOH-terminal sequence of MEKK2 (49) as described previously (74). Rabbit antiserum B130 was raised against a purified recombinant GST fusion protein containing amino acids 1-301 of MEKK3 (49).
Western Blot AnalysisProteins were separated by SDS-10% polyacrylamide gel electrophoresis and transferred onto a nitro-cellulose membrane for 40 min at 12 V in transfer buffer (48 mM Tris-HCl, 39 mM glycine, 1.3 mM SDS, 20% methanol) using a semidry transfer apparatus (Bio-Rad). Membranes were blocked for 1 h at room temperature with 5% nonfat milk powder in TTBS (50 mM Tris-HCl, pH 8.0, 0.1% Tween-20, 150 mM NaCl) and incubated for 1 h at room temperature or overnight at 4 °C with primary antibody (anti-Flag M2, 1:1000; anti-HA 12CA5, 1:1000; B128, 1:500; B130, 1:10,000) in TTBS containing 1% nonfat milk. After washing in TTBS, blots were incubated for 1 h at room temperature with the appropriate secondary antiserum (either goat anti-mouse or goat anti-rabbit IgG coupled to peroxidase (Sigma) at 1:1000) in TTBS containing 1% nonfat milk. Immunoblots were developed using enhanced chemiluminescence detection reagents (Amersham International).
Epitope-tagged MKK3 and MKK4 were separately expressed in
COS-7 cells either alone or in the presence of MEKK2 or MEKK3. MKK3 and
MKK4 were then isolated by immunoprecipitation, and their protein
kinase activities were assessed in vitro. Fig.
1A shows that MKK4 autophosphorylation was
enhanced by coexpression with either MEKK2 or MEKK3. MKK3 autocatalytic
activity was also enhanced by coexpression with MEKK3, whereas MEKK2
failed to cause a significant increase in MKK3 autophosphorylation
(Fig. 1A). MKK3 and MKK4 activity was also directly assessed
in the immune complex kinase assay using purified recombinant p38 or
JNK1 as substrates, respectively. Fig. 1B indicates that
MKK4-catalyzed phosphorylation of JNK1 was enhanced by coexpression of
MKK4 with either MEKK2 or MEKK3. By contrast, phosphorylation of p38 by
MKK3 was only stimulated following coexpression of MKK3 with MEKK3.
Western blot analysis using a monoclonal antiserum to the Flag epitope
showed that expression of MKK3 or MKK4 was essentially unaffected by
MEKK coexpression (Fig. 1C).
To determine whether MKK-catalyzed phosphorylation of JNK1 and p38
resulted in their activation, immune complex kinase assays of MKK3 and
MKK4 activity were performed in which ATF2 was provided as an in
vitro substrate for JNK1 or p38. Fig. 2A
shows that ATF2 phosphorylation by JNK1 was stimulated by
immunoprecipitates of MKK4 obtained from COS-7 cells coexpressing
either MEKK2 or MEKK3. Similar data were obtained in parallel
experiments in which p38 and ATF2 were the sequential substrates (Fig.
2B), confirming that p38 is also an effective substrate for
MKK4 in vitro (32, 37). Thus, MEKK2 and MEKK3 activated
MKK4, enhancing its ability to autophosphorylate and to phosphorylate
and activate JNK1 and p38 in vitro.
Activation of p38 by immuonprecipitated MKK3 was also observed following coexpression of MKK3 with MEKK3, whereas MEKK2 failed to activate the MKK3-p38 pathway (Fig. 2C). Of note, MKK3 activation could not be measured using JNK1 as substrate (data not shown), confirming that MKK3 is specific for p38 (32-35). Thus, by all criteria used to assess MKK3 activity (i.e. autophosphorylation, phosphorylation, and activation of p38), MKK3 is activated by MEKK3 but not by MEKK2.
MEKK2 Directly Activates MKK4 but Not MKK3The cotransfection
studies show that MKK4 is activated by MEKK2 and MEKK3, whereas MKK3
can only be activated by MEKK3. To establish whether these effects are
direct, epitope-tagged MEKK2 and MEKK3 were separately expressed in
COS-7 cells and isolated by immunoprecipitation. MEKK activity was then
assessed in an immune complex kinase assay using purified recombinant
MKK3 or MKK4 as substrates. Immunoprecipitated MEKK2 strongly
autophosphorylated in vitro, whereas MEKK3 did not (Fig.
3). This was not due to differences in MEKK expression
or recovery after immunoprecipitation, both of which are quantitatively
similar (data not shown). Immunoprecipitated MEKK2 caused a small but
equivalent increase in MKK3 and MKK4 phosphorylation, whereas MEKK3 was
without effect. In other experiments, MKK4 phosphorylation by MEKK2
exceeded that of MKK3 (not shown). Under different in vitro
conditions, the murine homologue of human MKK4, termed SEK1 or JNKK,
has been shown to be a good substrate for direct phosphorylation by
MEKK2 (49).
To reconstitute each activation pathway in vitro using recombinant proteins, JNK and ATF2 were provided with MKK4, whereas p38 and ATF2 were provided with MKK3 (Fig. 3). Immunoprecipitated MEKK2 activated MKK4, resulting in phosphorylation of ATF2 by JNK. Omission of MKK4 prevented ATF2 phosphorylation (data not shown), indicating that activation of MKK4 by MEKK2 was direct. In this and other experiments, MEKK2 caused little or no activation of MKK3, as shown by p38-dependent ATF2 phosphorylation. Thus, MEKK2 preferentially activates MKK4 over MKK3 in vivo (Figs. 1 and 2) and in vitro (Fig. 3). By contrast, immunoprecipitated MEKK3 was inactive with either MKK3 or MKK4 as substrate under all in vitro conditions examined. As has been speculated (49), the failure of MEKK3 to show significant kinase activity in vitro may be due to nonoptimal assay conditions or to loss of an essential cofactor during immunoprecipitation. Therefore, it is not possible at present to establish whether the activation of MKK3 by MEKK3 observed in vivo is direct.
Identification of the Sites of Activating Phosphorylation in MKK3 and MKK4MEKs are activated by phosphorylation on two serine residues within a regulatory region between kinase subdomains VII and VIII (38-41). These sites are necessary for MEK activation by Raf-1 and MEKK1 in vitro and in vivo. Sequence comparison of MKK3, MKK4, and MKK6 with MEK1 and MEK2 indicates that these phosphorylation sites are conserved (32-35). To test whether MKK3 and MKK4 activation by MEKK2 and MEKK3 required phosphorylation at these sites, a series of epitope-tagged MKK mutants were prepared in which one or both sites were replaced by alanine. Wild-type and mutant MKKs were separately expressed in COS-7 cells either alone or in the presence of MEKK2 or MEKK3. MKKs were then isolated by immunoprecipitation, and their protein kinase activities were measured in vitro.
Replacement of Ser189 and/or Thr193 in MKK3
with Ala blocked MEKK3-induced autophosphorylation (Fig.
4A) and activation of MKK3, as assessed by
its ability to phosphorylate and activate p38 using ATF2 as substrate
(Fig. 4B). Immunoblot analysis showed that MEKK3 and MKK3
mutants were each present at similar levels in the appropriate cell
lysates (Fig. 4C). These data indicate that both
Ser189 and Thr193 in MKK3 are essential for
MKK3 activation by MEKK3.
In the absence of MEKK coexpression, wild-type MKK4 displayed a low
basal autocatalytic activity that stimulated JNK1 in vitro (Figs. 5 and 6). This was blocked by the
substitution in MKK4 of Ser221 with Ala and greatly
attenuated by replacement of Thr225 with Ala. Coexpression
of these mutants with MEKK2 (Fig. 5) and MEKK3 (Fig. 6) demonstrated
that Ser221 was essential for autophosphorylation and
activation of MKK4. Although Thr225 in MKK4 was also
necessary for full activation by MEKK2 and MEKK3, a low level of MKK4
activation was observed when this site was replaced by Ala;
substitution of both Ser221 and Thr225 with Ala
blocked MEKK-induced activation of MKK4 (Figs. 5 and 6). Western blot
analysis using selective antisera indicated that the expression level
of both MEKK2 (Fig. 5C) and MEKK3 (Fig. 6C) in
the appropriate cell lysates was constant. Antiserum to the Flag
epitope also showed that wild-type and mutant forms of MKK3 or MKK4
were present at comparable levels in either the absence or presence of
coexpressed MEKK (Figs. 5C and 6C). These data, therefore, identify Ser221 and, to a lesser extent,
Thr225 in MKK4 as necessary sites for basal and
MEKK-induced autophosphorylation and activation of MKK4.
MEKK2 and MEKK3 Activate JNK1 but Fail to Activate p38 in Vivo
Consistent with the ability of MEKK2 and MEKK3 to activate
MKK4 in vivo, coexpression of either MEKK2 or MEKK3 in
HEK293 cells with epitope-tagged JNK1 resulted in comparable levels of
JNK1 activation when either ATF2 or c-Jun was provided as substrate (Fig. 7). Previous work has also shown that MEKK2 and
MEKK3 activate endogenous JNK in HEK293 cells but have no effect on
endogenous p38 activity (49). To extend these observations,
epitope-tagged p38 was expressed in HEK293 cells, isolated by
immunoprecipitation, and assayed for in vitro kinase
activity using ATF2 as substrate. Unlike JNK1, p38 autophosphorylated
under these conditions (Fig. 8). Treatment of cells with
anisomycin (Fig. 8) or sorbitol (not shown) activated p38, indicating
that the epitope-tagged enzyme was functionally coupled. However,
coexpression of p38 with either MEKK2 or MEKK3 failed to cause any
detectable p38 activation, whereas MKK3 expression produced a small
stimulatory effect on p38 activity. We surmised that the lack of effect
of MEKK3 on p38 activity was due to the absence of an MKK3-type enzyme
in HEK293 cells and that anisomycin-stimulated p38 activity was due to
an MKK3-independent pathway. We therefore coexpressed MEKK2 or MEKK3
together with MKK3 and p38 in these cells to reconstitute the pathway
in vivo. Surprisingly, under these conditions where MKK3 is
activated by MEKK3 (Figs. 1, 2, and 4), MEKK3 did not cause activation
of p38 (Fig. 8). These data have been reproduced using transient
expression in COS-7 cells (not shown) and suggest that regulation of
p38 and JNK1 by MEKK3 involves a mechanism to prevent p38 activation
but to allow JNK1 activation. The stress-activated pathways regulated
by MEKK2 and MEKK3 are summarized schematically in Fig.
9.
The work of Johnson and colleagues (49, 50) has identified three structurally and functionally related mammalian MEK kinase isozymes whose activities are clearly distinct from those of Raf kinases. Transient overexpression studies indicate that MEK kinases can regulate at least two separate MAPK signaling pathways involving the ERK and JNK subgroups (37, 41, 49-53). Although several of these reports suggest that MEKK1 activates MKK4 and/or JNK in preference to MEK and/or ERK (48, 52, 53), MEKK2 and MEKK3 do not appear to show a significant preference for activation of JNK relative to ERK (49). By contrast, Raf-1 is a specific activator of the ERK pathway and does not effect JNK activation (37, 52). This selectivity is attributed to the observation that the specific ERK activators, MEK1 and MEK2, are phosphorylated and activated by Raf-1 (38-41), whereas the JNK activator, MKK4, is not a substrate for Raf-1 (37).
Although a weak effect of MEKK1 on p38 activity in HEK293 cells has recently been reported (53, 64), attempts to demonstrate significant activation of p38 kinase by MEKK1, MEKK2, or MEKK3 have not been successful (37, 49). Since MKK4 can phosphorylate and activate JNK and p38 (32, 34, 37), it would not be predicted that MEK kinase isozymes that activate the MKK4/JNK cascade fail to stimulate p38 activity in the same cell type. However, the recent identification of MKK3 and MKK6 as specific activators of p38 (32-35) allows for a more direct assessment of MEKK involvement in the p38 activation pathway. In the present study, cotransfection experiments were performed to examine the regulation by MEKK2 and MEKK3 of the stress-activated protein kinase pathways involving p38 and JNK1.
We demonstrate here that MEKK2 and MEKK3 activate the MKK4/JNK pathway in vivo and that Ser221 and, to a lesser extent, Thr225 located in the conserved regulatory region of MKK4 are the critical sites for activating phosphorylation. Thus, replacement of Ser221 with Ala is sufficient to block autophosphorylation and activation of MKK4, whereas this substitution at Thr225 greatly attenuates MKK4 activity; substitution of both Ser221 and Thr225 with Ala blocks MEKK2- and MEKK3-induced activation of MKK4. A mutant of murine SEK1 lacking both phosphorylation sites equivalent to those in human MKK4 identified in this study is similarly not a substrate for MEKK1 in vitro and blocks activation of JNK when coexpressed with MEKK1 in L929 cells (48). The differential effects of Ser221 and Thr225 phosphorylation on MKK4 activity observed here have not previously been described. However, analogous single mutations in MEK1 suggest that phosphorylation of either Ser218 or Ser222 by Raf-1 is sufficient to cause MEK1 activation (40, 41). Raf-1 appears to phosphorylate both sites approximately equally, whereas MEKK1 shows a preference for Ser218 in MEK1 (41).
We also show in this report that MEKK3 activates MKK3 in vivo and that Ser189 and Thr193 in MKK3 are essential activating phosphorylation sites; substitution of one of these sites with Ala is sufficient to completely block MKK3 activation by MEKK3. Furthermore, MKK3 is not significantly activated by MEKK2 in vivo or in vitro, indicating that MKK3 is differentially regulated by MEKK2 and MEKK3. A recent report has demonstrated that MEKK1 can activate MKK4 but not MKK3 or MKK6 when their corresponding cDNAs are expressed in COS-7 cells (34). These studies, therefore, indicate that MKK3 is regulated by MEKK3 but not by MEKK1 or MEKK2. Since MEKK3 is inactive when isolated by immunoprecipitation (Fig. 3 and Ref. 49), it is not possible to determine at present whether MEKK3 directly phosphorylates and activates MKK3.
MKK3 and MKK6 have been established as specific physiologic regulators of p38 based on the following observations: MKK3 and MKK6 activate p38 in vitro (32, 33, 35); expression of an MKK3 or MKK6 mutant in which the dual activating phosphorylation sites have been replaced by acidic Glu residues causes constitutive p38 activation in vivo (33); expression of an MKK3 mutant in which Ser189 and Thr193 are replaced by Ala causes inhibition of UV-stimulated p38 activity (33). Therefore, the failure of MEKK3 to activate p38 when coexpressed in either the absence or presence of MKK3 is difficult to reconcile with our finding that MEKK3 clearly activates MKK3 in vivo. These observations are, however, strikingly similar to those reported by Xu et al. (51). In their study, both MEK1 and MEK2 were shown to be highly activated by cotransfection with MEKK1. Additionally, the catalytic domain of MEKK1 activated MEK1 and MEK2 in vitro and interacted with MEK in the yeast two-hybrid system. Despite these observations, MEKK1 failed to cause significant activation of endogenous or cotransfected ERK2, indicating that MEKs can be activated by MEKK1 without a concomitant activation of the ERK. The authors (51) speculate that MEKK1 expression may cause expression of an ERK phosphatase or make activated MEK inaccessible to the ERK substrate.
Very recently, Moriguchi et al. (35) have provided evidence
that MKK3 and MKK6 can be regulated by transforming growth
factor--activated kinase, or TAK-1, a newly described member of the
MEKK family. In addition, coexpression of TAK-1 and p38 in COS-7 cells
leads to activation of p38 (35). Both MEKK3 and TAK-1 are capable of
activating JNK, but, unlike TAK-1, MEKK3 also activates the ERK
signaling pathway (35, 49, 58). It is possible that the lack of
activation of p38 by cotransfected MEKK3 is due to coactivation of a
parallel MAPK signaling pathway, possibly involving ERK, which feeds
back to inhibit p38 kinase activity. In this regard, a number of dual
specificity MAPK phosphatases have been identified whose expression is
induced by cellular stress and/or mitogenic stimulation (75),
suggesting that these phosphatases play a role in the negative feedback
regulation of MAPKs. Such a mechanism has been described for ERK
regulation in NIH3T3 cells, where activation of JNK by cellular stress
or induction of MEKK1 expression leads to increased expression of MAPK
phosphatase-1 (76), a phosphatase that can dephosphorylate and
inactivate ERK. Although there is little information regarding the
substrate selectivity of different MAPK phosphatases (77), it seems
likely from the specific mechanisms by which each MAPK subgroup is
activated that complementary mechanisms exist to allow for their
differential inactivation.
Clearly, one limitation of the cotransfection approach that we have taken to study MAPK regulation is that it provides no information regarding the temporal relationship between MEKK expression and activation of individual signaling pathways involving ERK, JNK, and p38. It seems likely that longer term regulation of these pathways by MEKKs will also involve specific changes in expression of proteins (e.g. dual specificity MAPK phosphatases (75) and nonenzymatic MAPK inhibitors such as p21WAF1 (78, 79)) that serve to attenuate individual MAPK subtypes in a heterologous or homologous fashion. Based on the data described here, we speculate that rapid induction of MEKK3 expression in model cells will lead to transient p38 activation. If correct, then this system should also provide a model in which to examine the specific mechanisms involved in p38 inactivation.
We are grateful to Dr. R. J. Davis for the generous gift of MKK3, MKK4, p38, JNK1, ATF2, and Jun expression constructs. We also thank Dr. G. L. Johnson for providing HA-tagged JNK1 cDNA. We are also grateful to Benjamin Arrowsmith for excellent technical support and for preparation of MKK3 and MKK4 mutants.