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Originally published In Press as doi:10.1074/jbc.M705783200 on August 28, 2007

J. Biol. Chem., Vol. 282, Issue 42, 30476-30484, October 19, 2007
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MEKK4 Stimulation of p38 and JNK Activity Is Negatively Regulated by GSK3beta*

Amy N. Abell1, Deborah A. Granger, and Gary L. Johnson

From the Department of Pharmacology and the Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7365

Received for publication, July 13, 2007 , and in revised form, August 17, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The MAPK kinase kinase MEKK4 is required for neurulation and skeletal patterning during mouse development. MEKK4 phosphorylates and activates MKK4/MKK7 and MKK3/MKK6 leading to the activation of JNK and p38, respectively. MEKK4 is believed to be auto-inhibited, and its interaction with other proteins controls its dimerization and activation. TRAF4, GADD45, and Axin each bind and activate MEKK4, with TRAF4 and Axin binding to the kinase domain and GADD45 binding within the N-terminal regulatory domain. Here we show that similar to the interaction with TRAF4 and Axin, the kinase domain of MEKK4 interacts with the multifunctional serine/threonine kinase GSK3beta. GSK3beta binding to MEKK4 blocks MEKK4 dimerization that is required for MEKK4 activation, effectively inhibiting MEKK4 stimulation of the JNK and p38 MAPK pathways. Inhibition of GSK3beta kinase activity with SB216763 results in enhanced MEKK4 kinase activity and increased JNK and p38 activation, indicating that an active state of GSK3beta is required for binding and inhibition of MEKK4 dimerization. Furthermore, GSK3beta phosphorylates specific serines and threonines in the N terminus of MEKK4. Together, these findings demonstrate that GSK3beta binds to the kinase domain of MEKK4 and regulates MEKK4 dimerization. However, unlike TRAF4, Axin, and GADD45, GSK3beta inhibits MEKK4 activity and prevents its activation of JNK and p38. Thus, control of MEKK4 dimerization is regulated both positively and negatively by its interaction with specific proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MEKK4 is a 180-kDa mitogen-activated protein kinase kinase kinase (MAP3K)2 that functions in neurulation and skeletal patterning in the developing mouse embryo (1, 2). Mice harboring a kinase-inactive MEKK4 generated by knock-in mutation of the active site lysine K1361R are similar in phenotype to the knock-outs of TRAF4 and dishevelled-2 (1, 3, 4). TRAF4 and dishevelled-2 knock-out embryos and MEKK4 kinase-inactive knock-in embryos have significant defects in neural tube closure and skeletal patterning, including vertebral/rib malformations and scoliosis (3, 4). TRAF4 directly binds MEKK4 inducing MEKK4 dimerization and activation of MEKK4 kinase activity toward MKK4/7, resulting in the activation of JNK (5). Although the phenotypes of the dishevelled-2 knock-out and MEKK4 kinase-inactive knock-in are similar, there is no evidence that dishevelled-2 interacts with MEKK4. MEKK4 and dishevelled-2 are both binding partners of Axin, a scaffold protein involved in Wnt and Notch signaling (6). The binding of MEKK4 to Axin has been shown to regulate JNK activity (7), and here we also show that MEKK4 controls Axin-dependent activation of JNK and p38. Thus, dishevelled-2 and MEKK4 are both binding partners for Axin and genetically are in a common pathway based on their strongly overlapping phenotypes when deleted or mutated in the developing mouse embryo (1, 3, 6).

MEKK4 is predicted to exist in an auto-inhibited state with the N terminus folded over the C-terminal kinase domain (8). Binding to MEKK4 by activators, including TRAF4, GADD45{alpha}, -beta, and -{gamma}, and possibly Axin is thought to release MEKK4 from its auto-inhibited state and to promote MEKK4 homodimerization (5, 8, 9). The requirement of MEKK4 dimerization in promoting activation was demonstrated using a chemical inducer of dimerization to activate MEKK4 (5). The fact that MEKK4 and dishevelled-2 both bind Axin and appear genetically to be in the same pathway led us to search for other Axin-interacting proteins that would potentially regulate MEKK4 activity. Analysis of the MEKK4 primary sequence identified several putative GSK3 phosphorylation sites.

GSK3 is a serine/threonine kinase that regulates multiple signaling pathways. There are two highly homologous isoforms, GSK3{alpha} and GSK3beta. GSK3beta is a constitutively active kinase that is regulated by phosphorylation, localization, and GSK3beta-binding proteins (10, 11). GSK3beta is activated by phosphorylation of tyrosine 216 and negatively regulated by phosphorylation of serine 9 (10, 11). GSK3beta is a negative regulator of several signaling networks in cells. For example, GSK3 inhibits the canonical Wnt signaling pathway by promoting beta-catenin degradation (10–12). In addition, GSK3beta has been shown to negatively regulate JNK activation, but the mechanism of this regulation is undefined (13). Here we show that GSK3beta binds the kinase domain of MEKK4, inhibiting dimerization and MEKK4 activity. The findings define MEKK4 as a MAP3K regulated by GSK3beta for the negative regulation of JNK and p38 signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines, Culture Conditions, and Transfections—COS-7 cells were cultured in Dulbecco's modified Eagle's high glucose media supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin. Transfection of COS-7 cells was performed in 35- and 60-mm dishes for 24 h using Lipofectamine Plus (Invitrogen) according to the manufacturer's specifications. Trophoblast stem (TS) cells were isolated from blastocysts as described previously (14). Blastocysts were isolated at E3.5 from timed matings of 129 SvEv mice according to university and federal guidelines for the use of animals. TS cells were cultured without feeders in 30% TS media (RPMI 1640, 20% heat-inactivated fetal bovine serum, 1% sodium pyruvate, 1% penicillin and streptomycin, 1% glutamine, and 100 µM beta-mercaptoethanol) and 70% conditioned media isolated from primary mouse embryonic fibroblasts (MEFs) cultured in TS media. Isolation of MEFs from littermate wild-type and homozygous MEKK4K1361R MEFs was described previously (1). For treatment with GSK3 inhibitor, cells were incubated with 30 µM SB216763 (Sigma).

Plasmids—Wild-type and K1361R FLAG-tagged MEKK4 were constructed as described previously (5). Full-length HA-tagged MEKK4 and HA-tagged MEKK4 kinase domain were as described previously (15). HA-tagged GSK3beta wild-type and K85M constructs were kind gifts of Xianjun Fang (Virginia Commonwealth University). Potential serine/threonine phosphorylation sites were mutated to alanines using the QuikChange® multisite-directed mutagenesis kit (Stratagene). The resulting four mutated fragments were verified by sequencing and reinserted into full-length FLAG-tagged MEKK4.

Immunoprecipitations, Western Blot Analysis, Measurement of JNK and p38 Activity, and Kinase Assays—For kinase assays using purified proteins, His-MEKK4 purified from Sf9 cells was incubated with His-GSK3beta (Sigma) in kinase buffer containing 20 mM HEPES, pH 7.4, 10 mM MgCl2, 1 mM dithiothreitol, 0.1 mM sodium vanadate, 10 mM beta-glycerophosphate, and 10 mCi of [{gamma}-32P]ATP for 20 min at 30 °C. Proteins were separated by SDS-PAGE, visualized by autoradiography, and quantitated using a PhosphorImager. For assays with immunoprecipitated proteins, cells were lysed in cold buffer A containing 20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 1 mM sodium fluoride, 0.05 mM dithiothreitol, 1 µg/ml leupeptin, and 17 µg/ml aprotinin as described previously (1, 5). All chemicals were from Sigma. Lysates were immunoprecipitated, and blots were probed with anti-MEKK4 (1, 5), anti-FLAG (Sigma), anti-HA (12CA5), anti-GSK3beta (Santa Cruz Biotechnology), anti-TRAF4 (Santa Cruz Biotechnology) antibodies, and phospho-specific antibodies, including anti-phospho-MKK3/6, anti-phospho-JNK, and anti-phospho-p38 (Cell Signaling). Kinase assays were performed as described previously (1). Briefly, immunoprecipitates were washed with cold kinase buffer described above. Reactions were incubated with kinase buffer containing kinase-inactive His-MKK6 and cold 0.5 mM ATP or 10 mCi of [{gamma}-32P]ATP for 20 min at 30 °C. Cold kinase assays were probed with anti-phospho-MKK3/6 (Cell Signaling), and radioactive assays were exposed to film and quantitated with a PhosphorImager.

{lambda} Protein Phosphatase Treatment—2.5 µg of His-MEKK4 purified from Sf9 cells or immunoprecipitated HA-MEKK4 from transfected COS-7 cells was incubated for 45 min at 30 °C in the absence or presence of 800 units of {lambda} protein phosphatase (New England Biolabs). Phosphatase reactions were terminated by washing twice with cold buffer A containing phosphatase inhibitors and once with kinase buffer described above. Reactions were incubated with or without His-GSK3beta in radioactive kinase buffer as described above.


Figure 1
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  		FIGURE 1.
Phosphorylation of His-MEKK4 by His-GSK3beta invitro.A, the N terminus of MEKK4 contains three putative GSK3beta consensus sites. Consensus sequences were identified with in silico analysis using Scansite software. An underline indicates the serines and/or threonines predicted to be phosphorylated by GSK3beta. B, purified recombinant His-GSK3beta phosphorylates purified His-MEKK4. His-tagged MEKK4 purified from Sf9 cells was incubated in the absence or presence of increasing units of His-GSK3beta. Purified proteins were incubated in kinase buffer with [{gamma}-32P]ATP, and [{gamma}-32P]ATP-labeled proteins were visualized by autoradiography and quantitated by PhosphorImager. Fold increases in the phosphorylation of His-MEKK4 obtained from two independent experiments expressed as the mean ± range were as follows: 2 units, 25.4 ± 6.1; 4 units, 32.1 ± 5.5; 10 units, 52.5 ± 28.8. C, immunoprecipitated (IP) HA-MEKK4 is phosphorylated by purified His-GSK3beta. Lysates from COS-7 cells transfected with either wild-type or kinase-inactive (K1361M) HA-MEKK4 were immunoprecipitated with anti-HA antibody. Immunoprecipitates were incubated with 4 units of purified His-GSK3beta in kinase buffer with [{gamma}-32P]ATP, and [{gamma}-32P]ATP-labeled proteins were visualized by autoradiography (Autorad) and quantitated by PhosphorImager. HA-MEKK4 expression in immunoprecipitates was monitored by anti-HA Western blotting. A representative experiment from two independent experiments with similar results is shown. D, GSK3beta phosphorylation of His-MEKK4 is prevented by {lambda}-phosphatase pretreatment of MEKK4. His-MEKK4 was treated with buffer alone or 800 units of {lambda}-phosphatase for 45 min at 30 °C. Beads were washed and incubated in kinase buffer with [{gamma}-32P]ATP and with 5 units of His-GSK3beta as indicated. [{gamma}-32P]ATP-labeled proteins were visualized as in A. A representative experiment from three independent experiments with similar results is shown.

 


Figure 2
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  		FIGURE 2.
GSK3beta phosphorylates the N terminus of MEKK4. A, GSK3beta phosphorylates MEKK4, and this phosphorylation is independent of MEKK4 kinase activity. COS-7 cells were transfected with HA-MEKK4wt or HA-MEKK4K1361M in the absence or presence of HA-GSK3beta. HA-MEKK4 and HA-GSK3beta were immunoprecipitated (IP) together with an anti-HA antibody, and immunoprecipitates were incubated for 20 min at 30 °C with [{gamma}-32P]ATP and purified His-MKK6. An autoradiogram shows the phosphorylation of MEKK4 is increased greater than 20-fold in the presence of HA-GSK3beta. Because HA-GSK3beta and His-MKK6 migrate very closely, phosphorylation of His-MKK6 was monitored using a phospho-specific antibody for MKK6. Equal expression of HA-MEKK4 and HA-GSK3beta was monitored by Western blot with an anti-HA antibody. A representative experiment from three independent experiments with similar results is shown. B, GSK3beta phosphorylates the N terminus of MEKK4. Cells were transfected with FLAG-MEKK4wt, the N terminus of MEKK4, or HA-MEKK4 kinase domain in the absence or presence of GSK3beta. Lysates were immunoprecipitated with both anti-HA and anti-MEKK4 antibodies, and immunoprecipitates were incubated 20 min at 30 °C with [{gamma}-32P]ATP. An autoradiogram shows the phosphorylation of full-length MEKK4, N terminus of MEKK4, and kinase domain alone of MEKK4. A representative experiment from four independent experiments with similar results is shown. C, GSK3beta does not phosphorylate the kinase domain of MEKK4. COS-7 cells were transfected with full-length HA-MEKK4wt, wild-type HA-MEKK4wt kinase domain, or HA-MEKK4K1361R kinase domain in the absence or presence of HA-GSK3beta. Lysates were immunoprecipitated with anti-HA antibody, and immunoprecipitates were incubated as described in B. An autoradiogram shows the phosphorylation of full-length MEKK4, GSK3beta, and MEKK4 kinase domain. Western blotting with an anti-HA antibody shows equal protein expression. A representative experiment from two independent experiments with similar results is shown. D, phosphorylation of MEKK4 requires GSK3beta kinase activity. COS-7 cells were transfected with HA-MEKK4wt in the absence or presence of HA-GSK3betawt or HA-GSK3betaK85M. Lysates were immunoprecipitated with anti-HA antibody, and immunoprecipitates were incubated as described in B. An autoradiogram shows the phosphorylation of MEKK4. Western blotting with an anti-HA antibody shows equal protein expression. A representative experiment from three independent experiments with similar results is shown.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
GSK3beta Phosphorylates MEKK4—The consensus sequence for GSK3beta phosphorylation is (S/T)XXX(S/T)P (Fig. 1A) (11). The first serine or threonine in the consensus sequence is predicted to be phosphorylated by GSK3beta. Although not absolutely required, GSK3beta prefers substrates that are already phosphorylated on the second serine or threonine in the consensus sequence. This priming phosphorylation located four residues C-terminal to the GSK3beta phosphorylation site is thought to increase the efficiency of GSK3beta toward its substrates (11). Scanning of the MEKK4 sequence identified three consensus GSK3beta phosphorylation sequences (Fig. 1A). All of the consensus sequences were located in the N terminus of MEKK4 (amino acids 1–1326), whereas none were found in the catalytic domain (amino acids 1327–1597). Two of the potential sites, Ser77 and Thr112, are located approximately 50 residues upstream of the GADD45 binding domain. The third site, Ser1237, is located within a putative dimerization domain located 90 residues upstream of the kinase domain (9). To test the hypothesis that MEKK4 was a substrate for GSK3beta, purified recombinant GSK3beta and MEKK4 were used. MEKK4 purified from Sf9 cells was incubated with increasing units of recombinant GSK3beta. In vitro kinase assays were performed, and phosphorylation of MEKK4 was quantitated (Fig. 1B). Addition of 2 units of GSK3beta resulted in a 31-fold increase in MEKK4 phosphorylation demonstrating that MEKK4 is a direct phosphorylation substrate for GSK3beta. Similar results were obtained using immunoprecipitated HA-MEKK4 as a substrate for purified GSK3beta (Fig. 1C). Kinase-inactive MEKK4 (HA-MEKK4K1361M) was also a phosphorylation substrate for GSK3beta (Fig. 1C). However, the autophosphorylation of GSK3beta was not affected by the expression of either wild-type or kinase-inactive MEKK4K1361M (Fig. 1C).

As described above, preferred substrates for GSK3beta are generally primed by phosphorylation of a second site by another kinase (11). To test for a role of a second site priming phosphorylation on MEKK4, purified MEKK4 was treated with {lambda}-phosphatase, a protein with activity toward phosphorylated serine, threonine, and tyrosine (Fig. 1D). Subsequent GSK3beta in vitro kinase assays with control or phosphatase-treated MEKK4 demonstrated that phosphatase-treated MEKK4 was no longer a substrate for GSK3beta, consistent with the known properties of previously defined GSK3beta substrates (11). Identical results were obtained using {lambda}-phosphatase-treated immunoprecipitated HA-MEKK4 (data not shown). Cumulatively, the findings in Fig. 1 demonstrate that MEKK4 is a prototypical in vitro GSK3beta phosphorylation substrate.

When expressed alone, transfected MEKK4 exhibits a modest basal level of phosphorylation and kinase activity (Fig. 2A), as predicted from studies suggesting that the N terminus of MEKK4 is auto-inhibitory (9). In vitro kinase assay of coimmunoprecipitated GSK3beta and MEKK4 from cell lysates resulted in a 21-fold increase in the GSK3beta-dependent phosphorylation of MEKK4 compared with immunoprecipitation of MEKK4 in the absence of GSK3beta (Fig. 2A). The addition of purified, recombinant MKK6, a MEKK4 substrate (16), to the in vitro kinase assay resulted in the expected MEKK4-dependent phosphorylation of MKK6. Strikingly, coimmunoprecipitation of GSK3beta with MEKK4 resulted in a marked inhibition of MKK6 phosphorylation (Fig. 2A). This phosphorylation of MKK6 is MEKK4-dependent as kinase-inactive MEKK4K1361M that is phosphorylated by GSK3beta was unable to phosphorylate MKK6 (Fig. 2A).

Full-length MEKK4, the N terminus of MEKK4 upstream of the kinase domain, or the kinase domain of MEKK4 was expressed in the absence or presence of GSK3beta (Fig. 2B). Phosphorylation of full-length MEKK4 and the N terminus of MEKK4 in the presence of GSK3beta was similar, suggesting that GSK3beta phosphorylates the N terminus of MEKK4 (Fig. 2B). The kinase domain of MEKK4 is weakly autophosphorylated (Fig. 2, B and C), whereas the autophosphorylation of the kinase-inactive MEKK4K1361R kinase domain is completely inhibited (Fig. 2C). In the presence of GSK3beta, autophosphorylation of the kinase domain of MEKK4 was significantly reduced (Fig. 2, B and C), even though the MEKK4 kinase domain does not appear to be a phosphorylation substrate for GSK3beta (Fig. 2, B and C). Consistent with this conclusion, a downward mobility shift during SDS-PAGE of the kinase domain of MEKK4 was observed in the presence of GSK3beta, suggesting that GSK3beta inhibited the autophosphorylation of MEKK4. Fig. 2D shows that kinase-inactive GSK3beta (GSK3betaK85M) does not phosphorylate MEKK4, confirming that GSK3beta kinase activity is essential for phosphorylation of MEKK4.

Identification of MEKK4 Phosphorylation Sites for GSK3beta—Fig. 1A identified three consensus sequences in MEKK4 as potential sites for GSK3beta phosphorylation. Phosphatase treatment of MEKK4 inhibited GSK3beta phosphorylation of MEKK4, suggesting a priming phosphorylation is required for GSK3beta phosphorylation of MEKK4 (Fig. 1D). Both the consensus GSK3beta phosphorylation site and the priming site were mutated in each of the three consensus sequences (Fig. 3A). Mutation of each GSK3beta phosphorylation consensus sequence individually resulted in a partial loss of GSK3beta-catalyzed phosphorylation of MEKK4 (Fig. 3B). Mutation of serine 1237 and 1241 in the third consensus sequence resulted in the greatest inhibition of MEKK4 phosphorylation (Fig. 3B). Mutation of multiple consensus sequences was generally additive with simultaneous mutation of all three sequences resulting in a very dramatic loss of MEKK4 phosphorylation catalyzed by GSK3beta (Fig. 3C). These findings define three different sites of MEKK4 phosphorylation catalyzed by GSK3beta.

Endogenous GSK3beta and MEKK4 Are Binding Partners—Cumulatively, the findings in Figs. 1, 2, 3 clearly demonstrate that MEKK4 is a GSK3beta phosphorylation substrate. The findings in Fig. 2, A–C, and Fig. 3 are striking but seemed at first to be somewhat contradictory. First, GSK3beta phosphorylates three sites N-terminal to the kinase domain and inhibits MEKK4 phosphorylation of MKK6. Second, GSK3beta appears to inhibit the autophosphorylation activity of the MEKK4 kinase domain even though the MEKK4 kinase domain is not phosphorylated by GSK3beta. For these reasons, the interaction of endogenous MEKK4 and GSK3beta was examined by coimmunoprecipitation (Fig. 4A). Unlike GSK3beta that is expressed ubiquitously and strongly in most cell types, MEKK4 is expressed rather weakly in most adult cell types (1, 17). However, MEKK4 is expressed strongly during development in both embryonic and extraembryonic cells (1, 2). Therefore, the association of endogenous GSK3beta and MEKK4 was examined using trophoblast stem (TS) cells, which are epithelial extraembryonic cells that differentiate to form the tissues of the placenta (14). Immunoprecipitation of TS cell lysates with anti-GSK3beta antibody resulted in the specific coimmunoprecipitation of MEKK4 with GSK3beta (Fig. 4A). MEKK4 did not coimmunoprecipitate with a nonspecific anti-rabbit antibody (Fig. 4A). These data show the specific association of endogenous GSK3beta and MEKK4 in TS cells.


Figure 3
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  		FIGURE 3.
Identification of specific residues in the N terminus of MEKK4 that are phosphorylated by GSK3beta. A, description of MEKK4 phosphorylation mutants. B, phosphorylation of MEKK4 is reduced by mutation of specific serine and threonines in the N terminus of MEKK4. HA-GSK3beta was transfected with either wild-type or phosphorylation mutant FLAG-MEKK4 constructs as indicated. HA-GSK3beta and FLAG-MEKK4 were immunoprecipitated (IP) together with anti-HA and anti-FLAG antibodies, and immunoprecipitates were incubated for 20 min with [{gamma}-32P]ATP. Autoradiograms (Autorad) show phosphorylation of MEKK4 and GSK3beta. Western blotting with anti-FLAG and anti-HA antibodies shows equal expression of FLAG-MEKK4 and HA-GSK3beta. Phosphorylation of MEKK4 was measured using a PhosphorImager. Percent phosphorylation of MEKK4 was calculated by dividing mutant MEKK4 phosphorylation in the presence of HA-GSK3beta by the phosphorylation of MEKK4wt in the presence of HA-GSK3beta. A representative experiment from two independent experiments with similar results is shown. C, substitution of all putative GSK3beta phosphorylation sites results in a 71% reduction in MEKK4 phosphorylation. Experiments were performed as described in B. The percent phosphorylation of MEKK4 phosphorylation mutants relative to wild-type MEKK4 obtained from two independent experiments expressed as the mean ± range for M1, M2, and M3 and obtained from three independent experiments expressed as the mean ± S.E. for M4 and M5 were the following: M1, 72.8 ± 2.1; M2, 82.9 ± 2.9; M3, 67.7 ± 13.1; M4, 46.9 ± 13.8; and M5, 22.6 ± 8.9.

 


Figure 4
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  		FIGURE 4.
The association of MEKK4 and GSK3beta. A, endogenous coimmunoprecipitation of MEKK4 and GSK3beta. Trophoblast stem cell lysates were immunoprecipitated (IP) with a rabbit anti-GSK3beta antibody or control anti-rabbit antibody and probed with anti-MEKK4 and anti-GSK3beta antibodies. A representative experiment from three independent experiments with similar results is shown. NS indicates a nonspecific band related to heavy chain. C indicates a control lane lacking lysate and containing buffer and anti-GSK3beta antibody. B, FLAG-MEKK4 coimmunoprecipitates with HA-GSK3beta. COS-7 cells were transfected with FLAG-MEKK4wt and HA-GSK3beta as indicated. Lysates were immunoprecipitated with an anti-HA antibody and probed with anti-FLAG and anti-HA antibodies as indicated. A representative experiment from two independent experiments with similar results is shown. C, N terminus of MEKK4 does not bind to HA-GSK3beta. COS-7 cells were transfected with FLAG-MEKK4wt, MEKK4 N terminus, or HA-GSK3beta as indicated. Lysates were immunoprecipitated with an anti-HA antibody, and probed with anti-MEKK4 and anti-HA antibodies as indicated. A representative experiment from two independent experiments with similar results is shown. D, HA-GSK3beta binds the kinase domain of MEKK4. COS-7 cells were transfected with wild-type HA-MEKK4 kinase domain and HA-GSK3beta as indicated. Lysates were immunoprecipitated with an anti-GSK3beta antibody and probed with an anti-HA antibody. A representative experiment from two independent experiments with similar results is shown. E, MEKK4 phosphorylation mutant (MEKK4M5) retains GSK3beta binding. Lysates prepared from COS-7 cells transfected with the indicated constructs were immunoprecipitated with an anti-HA antibody and probed with anti-FLAG and anti-HA antibodies. A representative experiment from three independent experiments with similar results is shown.

 
GSK3beta Binds the Kinase Domain of MEKK4—Fig. 4B shows that FLAG-MEKK4 coimmunoprecipitated with HA-GSK3beta. Although GSK3beta phosphorylates the N terminus of MEKK4, the recognition of the MEKK4 phosphorylation sites by GSK3beta within the N terminus of MEKK4 is not of sufficient affinity to allow coimmunoprecipitation of the two proteins (Fig. 4C). Instead, GSK3beta binds to the kinase domain of MEKK4, as the kinase domain of MEKK4 strongly coimmunoprecipitated with GSK3beta (Fig. 4D). Interestingly, the MEKK4 M5 mutant protein with alanine substitutions in each of the defined GSK3beta consensus sequences (Fig. 3) retained the ability to coimmunoprecipitate with GSK3beta (Fig. 4E). These findings demonstrate that endogenous MEKK4 and GSK3beta are in a coimmunoprecipitating complex and that the kinase domain of MEKK4 is the primary site for the stable interaction of GSK3beta and MEKK4.

MEKK4 Dimerization Is Disrupted by the Binding of GSK3beta—MEKK4 forms homodimers that have been shown to activate MEKK4 kinase activity (5, 9). TRAF4 and GADD45{alpha}, -beta, and -{gamma} have each been shown to induce MEKK4 dimerization resulting in increased MEKK4 kinase activity (5, 9). Dimerization induced by a chemical dimerizer leads to activation of MEKK4 and the stimulation of JNK, a downstream pathway controlled by MEKK4 (5). Coexpressed HA-MEKK4 and FLAG-MEKK4 form a stable association as measured by the coimmunoprecipitation of HA-MEKK4 with FLAG-MEKK4, consistent with an oligomeric MEKK4 protein complex (Fig. 5). Coexpression of GSK3beta with HA-MEKK4 and FLAG-MEKK4 completely abrogates this HA-MEKK4-FLAG-MEKK4 complex (Fig. 5). In addition, HA-GSK3beta is strongly coimmunoprecipitated with FLAG-MEKK4 using an anti-FLAG antibody (Fig. 5). These data clearly show that GSK3beta binds MEKK4 and blocks MEKK4 dimerization, which is required for MEKK4 activation (5, 9).

GSK3beta Inhibits the Kinase Activity of MEKK4—Coexpression of GSK3beta with full-length MEKK4 inhibits MEKK4 kinase activity (Fig. 2A and Fig. 6A). Although the phosphorylation sites for GSK3beta lie in the N terminus of MEKK4, GSK3beta retains the ability to inhibit the kinase activity of the MEKK4 kinase domain in the absence of the MEKK4 N terminus (Fig. 6A). These findings demonstrate that GSK3beta inhibits MEKK4 kinase activity through the direct binding to the kinase domain of MEKK4.


Figure 5
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  		FIGURE 5.
GSK3beta blocks MEKK4 dimerization. Lysates prepared from COS-7 cells transfected with the indicated constructs were immunoprecipitated (IP) with an anti-FLAG antibody and probed with anti-HA and anti-FLAG antibodies. Equal protein expression was monitored by Western blotting of total lysates with anti-FLAG and anti-HA antibodies. A representative experiment from three independent experiments with similar results is shown.

 
Activation of MEKK4 results in the activation of both the JNK and p38 MAPK pathways (1, 15). Consistent with the inhibition of MEKK4 kinase activity in the presence of GSK3beta, MEKK4 activation of both p38 (Fig. 6B) and JNK (Fig. 6C) is inhibited by GSK3beta. The MEKK4 M5 mutant with all three GSK3beta phosphorylation sites mutated retains the ability to activate both JNK and p38, and this activity is inhibited by GSK3beta (Fig. 6, D and E). This finding is consistent with the retention of GSK3beta binding to the MEKK4 M5 phosphorylation mutant (Fig. 4E).

Inhibition of GSK3 by SB216763 Enhances MEKK4 Activation of JNK and p38—For reasons that are unclear, mutation of the GSK3beta active site lysine (GSK3betaK85M) impedes the binding of GSK3beta with MEKK4 (not shown). For this reason, we tested whether an active site inhibitor of GSK3 (GSK{alpha} and -beta), SB216763 (18), would alter GSK3beta regulation of MEKK4 activity. Cells coexpressing HA-MEKK4 and HA-JNK that were treated with SB216763 exhibited enhanced JNK phosphorylation compared with cells treated with carrier alone (Fig. 7A). SB216763 had no effect on JNK activation by the related MAP3K MEKK1 (Fig. 7B). SB216763 treatment had a more modest effect on MEKK4 activation of p38, as compared with MEKK4 activation of JNK. MEKK4 activation of p38 was not enhanced with 2.5 h of SB216763 treatment. However, prolonged treatment for 16 h resulted in a 50% increase in MEKK4 activation of p38 (Fig. 7C).

Enhanced MEKK4 Kinase Activity with Inhibition of GSK3 by SB216763—Fig. 7D shows that continuous exposure of cells expressing wild-type HA-MEKK4 to SB216763 over a 16-h time period resulted in enhanced MEKK4 kinase activity. HA-MEKK4 immunoprecipitated from cells treated for increasing time with SB216763 was subjected to in vitro kinase assays using kinase-inactive MKK6 as a substrate. A time-dependent increase in MEKK4 kinase activity was observed with SB216763 treatment demonstrating that inhibition of GSK3 enhanced MEKK4 kinase activity (Fig. 7D). The activity measured in the in vitro kinase assay was dependent on MEKK4, because kinase-inactive HA-MEKK4K1361M did not have any kinase activity toward MKK6 in the assay (Fig. 7D). In addition to enhancing the kinase activity of overexpressed MEKK4, treatment with GSK3 inhibitor also increased the kinase activity of endogenous MEKK4. Endogenous MEKK4 immunoprecipitated from MEFs treated with SB216763 exhibited a 34 ± 0.02% increase in kinase activity relative to treatment with carrier alone. The activation of MEKK4 resulting from inhibition of GSK3 kinase activity further demonstrates that GSK3 is a negative regulator of MEKK4 signaling. The loss of GSK3beta-mediated inhibition of MEKK4 kinase activity with SB216763 treatment is consistent with the observation that kinase-inactive GSK3betaK85M binds poorly to MEKK4, suggesting that the interaction of GSK3beta with MEKK4 involves residues within the kinase domain of GSK3beta.

MEKK4 Mediates Axin-dependent Activation of JNK and p38—It has been demonstrated that TRAF4 and GADD45 proteins both activate MEKK4 (5, 9). Fig. 8A and the work of others (7) show that the scaffolding protein, Axin, binds the kinase domain of MEKK4. Kinase-inactive MEKK4 (MEKK4K1361R) strongly abrogates the activation of both JNK and p38 in response to Axin expression (Fig. 8, B and C). These results show that Axin binds the kinase domain of MEKK4 and are the first to show that a kinase-inactive full-length MEKK4 inhibits Axin-mediated activation of both JNK and p38, suggesting that MEKK4 regulates Axin-dependent control of JNK and p38. GSK3beta also binds Axin (19), demonstrating that MEKK4 and GSK3beta are not only in a complex in cells but also interact with a common scaffold protein.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown previously that chemical induction of MEKK4 dimerization activates MEKK4 kinase activity (5). In addition, we have shown that through its TRAF domain TRAF4 also induces oligomerization of MEKK4 and activates its kinase activity (5). Activation of MEKK4 by two independent mechanisms, the FKBP dimerization system and TRAF4-induced oligomerization, clearly shows that like other protein kinases MEKK4 is activated by oligomerization and transphosphorylation in the kinase domain (5). It has also been proposed that GADD45 proteins induce dimerization required for activation of MEKK4 (9). It is unclear exactly how Axin activates MEKK4, but Axin is a dimer and could potentially activate MEKK4 in a manner similar to TRAF4 and GADD45 proteins (7, 20). Whereas Axin and TRAF4 bind to the C-terminal kinase domain of MEKK4, GADD45 proteins bind to an N-terminal region; thus there are multiple mechanisms to regulate MEKK4 dimerization. Our current findings demonstrate that endogenous MEKK4 and GSK3beta are binding partners in cells, and like TRAF4 and Axin, GSK3beta binds to the C-terminal region that encodes the kinase domain of MEKK4. The binding of GSK3beta to the MEKK4 C-terminal kinase domain inhibits MEKK4 kinase activity by inhibiting MEKK4 dimerization. Fig. 9A shows a schematic model of the known effectors that control MEKK4 activity. It is interesting that multiple proteins that directly bind MEKK4 regulate its activity by controlling its dimerization.


Figure 6
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  		FIGURE 6.
GSK3beta inhibits MEKK4 signaling. A, GSK3beta inhibits the kinase activity of both full-length and kinase domain only MEKK4. Lysates prepared from COS-7 cells transfected with the indicated constructs were immunoprecipitated with an anti-HA antibody. Immunoprecipitates were subjected to a nonradioactive in vitro kinase activity with the substrate His-MKK6. Activity was measured as described in Fig. 2. The percent inhibition of MEKK4 full-length and kinase domain only activity by GSK3beta expression is 54 ± 15 and 68 ± 3, respectively. B, GSK3beta inhibits MEKK4 activation of p38. COS-7 cells were transfected with the indicated constructs. p38 activity was monitored using a phospho-specific anti-p38 antibody. Anti-HA and anti-FLAG antibodies were used to monitor protein expression levels. The percent inhibition of MEKK4 activation of p38 by GSK3beta expression is 38 ± 10. C, GSK3beta inhibits MEKK4 activation of JNK. COS-7 cells were transfected with the indicated constructs. JNK activity was monitored using a phospho-specific anti-JNK antibody. Anti-HA antibody was used to monitor protein expression levels. The percent inhibition of MEKK4 activation of JNK by GSK3beta expression is 48 ± 12. D, GSK3beta retains the ability to inhibit MEKK4 phosphorylation mutant activation of JNK. Cells were treated as in C. The percent inhibition of wild-type MEKK4 and M5 mutant activation of JNK by GSK3beta expression is 57 ± 15 and 52 ± 3.3, respectively. E, GSK3beta retains the ability to inhibit MEKK4 phosphorylation mutant activation of p38. Cells were treated as in B. The percent inhibition of wild-type MEKK4 and M5 mutant activation of p38 by GSK3beta expression is 28 ± 0.5 and 16 ± 24, respectively. A–E, a representative experiment from two independent experiments with similar results is shown.

 
GSK3beta has been shown previously to be a negative regulator of JNK signaling (13). However, the mechanisms whereby GSK3beta decreases JNK signaling were unknown. Here we show that GSK3beta inhibits MEKK4 kinase activity and MEKK4 activation of JNK. The exact mechanism of how GSK3beta inhibits MEKK4 activity is unclear, but several findings suggest that the active site of GSK3beta might bind MEKK4. The GSK3beta inhibitor, SB216763, binds to the active site of GSK3beta and blocks GSK3beta inhibition of MEKK4. Mutation of the kinase domain active site lysine in GSK3beta (K85M) causes a marked decrease in the stable interaction of GSK3beta and MEKK4, suggesting a functional GSK3beta active site is required for MEKK4 binding. This hypothesis is consistent with the finding that GSK3beta binding to the MEKK4 kinase domain inhibits MEKK4 kinase activity. However, the kinase domain of MEKK4 does not appear to be a substrate for GSK3beta-catalyzed phosphorylation, so the inhibition of MEKK4 kinase activity by GSK3beta appears to be directly due to protein-protein interaction and not phosphorylation. The phosphorylation of the MEKK4 N-terminal residues defined in Figs. 1, 2, 3 does not have a measurable function in controlling MEKK4 inhibition by GSK3beta, and so far we have been unable to define a function for GSK3beta phosphorylation of these sites in the MEKK4 N terminus. It is possible that phosphorylation of these sites controls the interaction of MEKK4 with other proteins such as GADD45 or Axin, but we have no evidence of this, and additional studies will be required to answer this difficult question. The MEKK4 M5 mutant with substitution of all three GSK3beta consensus phosphorylation sites still retains a low level of GSK3beta-dependent phosphorylation. The presence of this residual phosphorylation suggests that there may be additional GSK3beta phosphorylation sites in the N terminus of MEKK4. In addition, this residual phosphorylation of MEKK4 by GSK3beta may be sufficient for GSK3beta-dependent regulation of MEKK4 function by phosphorylation.

The finding that GSK3beta is a negative regulator of MEKK4 is of great importance in defining the function of MEKK4 in mouse development. MEKK4 binds Axin, TRAF4, and GSK3beta, and GSK3beta also binds Axin (5, 7, 19). Our observations indicate that TRAF4 and Axin are not binding partners.3 Therefore, there is a complex interaction of Axin, GSK3beta, MEKK4, and TRAF4, all of which are critical in early embryonic development (1, 2, 4, 21, 22), with MEKK4 acting as a signaling hub to positively control JNK and p38 activity in response to TRAF4 and Axin and negatively regulate these pathways in response to GSK3beta (Fig. 9B). In the connections map proposed in Fig. 9B, MEKK4 is a central MAP3K for the control of signaling in the Axin-regulated Wnt signaling pathway. In addition, literature searches show that DTRAF1, the Drosophila TRAF most similar to mammalian TRAF4, interacts with Misshapen, the mammalian homolog of Nck-interacting kinase (23), which indirectly may connect TRAF4 to dishevelled and Axin. Interestingly, dishevelled 1/2, Axin, TRAF4, and JNK1/2 gene knock-outs and the MEKK4 kinase-inactive gene knock-in all display similar neurulation defects during embryonic mouse development (1, 3, 4, 21, 24, 25). In addition, TRAF4, dishevelled 1/2, and conditional c-Jun knock-outs and the MEKK4 kinase-inactive knock-in also have skeletal malformations (1, 3, 4, 26). The GSK3beta knock-out is lethal between E13.5 and E14.5 because of liver apoptosis (22). Redundant signaling by GSK3{alpha} is thought to compensate for the loss of GSK3beta, except in the liver (22, 27). Loss of both GSK3{alpha} and GSK3beta is early embryonic lethal, so it is unclear how GSK3 function influences neurulation and vertebral/rib patterning (27). Therefore, our studies begin to provide a biochemical explanation for the genetic pathway that has been mapped by overlapping knock-out and knock-in phenotypes for MEKK4, TRAF4, disheveled 1/2, GSK3beta, and Axin. MEKK4 function is clearly critical in this signaling network, and its kinase activity is both positively and negatively regulated by proteins within the network for control of JNK and p38.


Figure 7
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  		FIGURE 7.
Inhibition of GSK3 with the GSK3 inhibitor SB216763 enhances MEKK4 activation of JNK. A, inhibition of GSK3 enhances MEKK4 activation of JNK. COS-7 cells transfected with the indicated constructs were treated for 2 h with carrier alone (Me2SO) or SB216763. Lysates were probed with either a phospho-specific anti-JNK antibody to measure JNK activity or an anti-HA antibody to measure total protein expression. A representative experiment from four independent experiments with similar results is shown. B, inhibition of GSK3 does not alter MEKK1 activation of JNK. COS-7 cells were transfected with HA-MEKK1, and cells were treated as described in A. A representative experiment from three independent experiments with similar results is shown. C, inhibition of GSK3 enhances MEKK4 activation of p38. COS-7 cells were transfected with the indicated constructs and treated as in A. Lysates were probed with a phospho-specific anti-p38 antibody to measure active p38 and an anti-HA antibody to detect HA-MEKK4 expression. A representative experiment from three independent experiments with similar results is shown. D, GSK3 inhibition with SB216763 increases MEKK4 kinase activity. COS-7 cells were transfected as indicated, and cells were treated for the indicated number of hours with either Me2SO or SB216763. HA-MEKK4 was immunoprecipitated with anti-HA antibody, and a radioactive kinase assay with kinase-inactive His-MKK6 as the substrate was performed. The incorporation of [{gamma}-32P]ATP into His-MKK6 is shown by autoradiogram and measured with a PhosphorImager. Equal immunoprecipitation of MEKK4 is shown by Western blotting with an anti-HA antibody. A representative experiment from three independent experiments with similar results is shown. The fold stimulation of MEKK4 kinase activity relative to empty vector alone expressed as the mean ± S.E. for the indicated treatments is as follows: 0h, 4.5 ± 0.5; 2h, 6.2 ± 1.2; 4h, 4.7; 16 h, 6.9 ± 0.4; kinase-dead MEKK4 at 0h, 1.1 ± 0.3.

 


Figure 8
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  		FIGURE 8.
Axin binds the kinase domain of MEKK4. A, Axin coimmunoprecipitates with the kinase domain of MEKK4. MEKK4 kinase domain was immunoprecipitated (IP) with an anti-MEKK4 antibody from lysates prepared from cells transfected with the indicated constructs. HA-Axin coimmunoprecipitating with MEKK4 kinase domain was detected with anti-HA antibody. Anti-HA antibody was used to monitor total protein expression. A representative experiment from four independent experiments with similar results is shown. B, kinase-inactive MEKK4K1361R inhibits Axin activation of JNK. COS-7 cells were cotransfected with HA-JNK, HA-Axin, and FLAG-MEKK4K1361R. Phosphorylation of HA-JNK was measured with anti-phospho-JNK antibody. Total protein was monitored with anti-HA and anti-FLAG antibodies. A representative experiment from two independent experiments with similar results is shown. C, kinase-inactive MEKK4K1361R inhibits Axin activation of p38. Phosphorylation of FLAG-p38 was measured with anti-phospho-p38 antibody. Total protein was monitored with anti-HA and anti-FLAG antibodies. A representative experiment from two independent experiments with similar results is shown.

 


Figure 9
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  		FIGURE 9.
Regulation of MEKK4 activation of JNK and p38. A, schematic diagram of the regulation of MEKK4 signaling to JNK and p38. GADD45 stimulates MEKK4 activation by binding the N terminus of MEKK4. TRAF4, Axin, and GSK3beta all bind to the kinase domain of MEKK4. Unlike TRAF4 and Axin that activate MEKK4, GSK3beta inhibits MEKK4 signaling to JNK and p38. The location of Ser77, Thr112, and Ser1237 that are phosphorylated by GSK3beta are indicated. The location and residues of the catalytic domain of MEKK4 are also indicated. B, proposed connections map showing MEKK4 as a signaling hub for the activation of JNK and p38. See text for discussion.

 

   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM30324. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Pharmacology, 1012 Mary Ellen Jones Bldg., CB 7365 Chapel Hill, NC 27599-7365. Tel.: 919-843-3257; Fax: 919-966-5640; E-mail: amy_abell{at}med.unc.edu.

2 The abbreviations used are: MAP3K, mitogen-activated protein kinase kinase kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; TS, trophoblast stem; MEF, mouse embryonic fibroblast; HA, hemagglutinin; E, embryonic day. Back

3 A. N. Abell, D. A. Granger, and G. L. Johnson, unpublished observations. Back



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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