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J. Biol. Chem., Vol. 281, Issue 10, 6236-6245, March 10, 2006
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1
From the
Department of Pharmacology and Toxicology, University of Arizona College of Pharmacy, Tucson, Arizona 85721 and the Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232
Received for publication, August 22, 2005 , and in revised form, December 26, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B kinase. Phosphorylation is believed to be a critical component for MEKK3-dependent signal transduction, but little is known about the phosphorylation sites of this MAP3K. To address this question, point mutations were introduced in the activation loop (T-loop), substituting alanine for serine or threonine, and the mutants were transfected into HEK293 Epstein-Barr virus nuclear antigen cells. MEKK3-dependent activation of an NF-B reporter gene as well as ERK, JNK, and p38 MAP kinases correlated with a requirement for serine at position 526. Constitutively active mutants of MEKK3, consisting of S526D and S526E, were capable of activating a NF-B luciferase reporter gene as well as ERK and MEK, suggesting that a negative charge at Ser526 was necessary for MEKK3 activity and implicating Ser526 as a phosphorylation site. An antibody was developed that specifically recognized phospho-Ser526 of MEKK3 but did not recognize the S526A point mutant. The catalytically inactive (K391M) mutant of MEKK3 was not phosphorylated at Ser526, indicating that phosphorylation of Ser526 occurs via autophosphorylation. Endogenous MEKK3 was phosphorylated on Ser526 in response to osmotic stress. In addition, phosphorylation of Ser526 was required for MKK6 phosphorylation in vitro, whereas dephosphorylation of Ser526 was mediated by protein phosphatase 2A and sensitive to okadaic acid and sodium fluoride. Finally, the association between MEKK3 and 14-3-3 was dependent on Ser526 and prevented dephosphorylation of Ser526. In summary, Ser526 of MEKK3 is an autophosphorylation site within the T-loop that is regulated by PP2A and 14-3-3 proteins. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Over the last decade, a large body of work has characterized events that occur downstream of the MAPKs. However, little is known regarding the regulatory mechanisms that modulate the MEKK proteins to ultimately regulate the MAPKs. For example, it is known that overexpression of MEKK3 activates the ERK (3, 4), JNK (3–5), p38 (5, 6), ERK5 (7), and NF-B pathways (8–10). Typically, MEKK3-dependent regulation of these pathways is studied by using transfection studies, and activation of these pathways rarely requires an agonist. Therefore, it appears that some process intrinsic to MEKK3 is critical and sufficient for activation of these pathways.
The activation loop of some protein kinase families, such as the arginine-aspartate family, is positioned between subdomains VII and VIII and is phosphorylated by other protein kinases or through autophosphorylation of the kinase itself (11). Phosphorylation within the activation loop of protein kinases results in conformational changes in the protein structure that (i) enhance substrate binding, (ii) correctly position amino acids involved in catalysis, and (iii) relieve steric hindrance within the catalytic domain. Regardless of how the activation loop is phosphorylated, regulation of catalytic activity frequently correlates with phosphorylation of the activation loop.
Given that phosphorylation plays a critical role in regulating the MAPKs and the MAP2Ks, we investigated how phosphorylation of MEKK3 might affect its catalytic activity. Since phosphorylation sites within the activation loop of MEKK3 have not been reported, we systematically mutated serine and threonine residues within the activation loop to alanine and monitored MEKK3-dependent activities in HEK293 EBNA cells. Two key amino acids were identified at positions 526 and 530 using a luciferase-based reporter gene assay as well as assays that measure the ERK, JNK, and p38 MAP kinases. We demonstrate that Ser526 is phosphorylated by using a specific antibody that recognizes phospho-Ser526. In contrast, Thr530 is not phosphorylated but is required for MEKK3 catalytic activity. In addition, phosphorylation of endogenous MEKK3 occurs on Ser526 in response to osmotic stress. Finally, dephosphorylation of phospho-Ser526 is mediated by PP2A, and the association of 14-3-3 with phosphorylated MEKK3 prevents dephosphorylation by PP2A.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The 14-3-3 antibody recognizes amino acids 3–21 of the human isoform of 14-3-3, and this sequence is highly conserved within the 14-3-3 family of proteins (, 
, 
, 
, 
, 
, and 
). An antibody to MEKK3 was generated as previously described (12). Monoclonal antibodies recognizing the hemagglutinin and the FLAG epitopes were obtained from Roche Applied Science. Anti-FLAG affinity resin was obtained from Sigma. Plasmids and Site-directed Mutagenesis—FLAG·MEKK3 was excised from pBTM116 using SalI and ligated into the EcoRV site of pcDNA3.1 HisA (12). FLAG·MEKK3 in pcDNA3.1 HisA was used as a template for PCR to generate 370-bp fragments containing point mutations in the activation loop, replacing serine or threonine with alanine, aspartic, or glutamic acid. The 370-bp fragment was flanked by HindIII and NotI endonuclease sites. Since the 5' SalI site was lost after ligation of FLAG·MEKK3 into pcDNA3.1 HisA, the NotI site of the polylinker was used as a 3' cloning site in subsequent steps.
The megaprimer method of site-directed mutagenesis, which involved two PCR steps, was utilized to generate point mutants. Unique sense primers, listed in Table 1, and the antisense primer, 5'-T ACC TAG CAG GAA CAG ATT GTG TCG ACC TCA GTA CAC T-3', were used in the first reaction (13). The product of the first reaction was used as a template for the second reaction with an MEKK3 sense primer, 5'-GGC ATG TCA TAC CTG CAC AGC-3', which is upstream of the HindIII site, and antisense primer 5'-T ACC TAG CAT GAA CAG ATT G-3'. The product of this reaction was subcloned into pGEM-T and sequenced to verify the nucleotide sequence. The 370-bp HindIII/NotI fragment containing the mutation was ligated with the 1800-bp KpnI/HindIII fragment of MEKK3 into pcDNA3.1 HisA that was cleaved with KpnI/NotI. The same procedure was used to construct the point mutants listed in Table 1.
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Cell Culture—HEK293 EBNA cells were maintained at 37 °C, 5% CO2, with Dulbecco's modified Eagle's medium, pH 7.4 (Invitrogen), supplemented with 10% fetal bovine serum, 250 µg/ml Geneticin, 100 units/ml penicillin, and 100 µg/ml streptomycin.
Transfections—HEK293 EBNA cells were transiently transfected with an empty vector as a control, MEKK3, K391M, or point mutants in the pcDNA3.1 HisA plasmid using 9 µl of Fugene 6 (Roche Applied Science) and 3 µg of DNA. Cells were transfected in OPTI-MEM serum-free medium (Invitrogen) and then replaced with complete medium 5 h post-transfection.
Immunoprecipitation and Immunoblotting—Cells were washed twice with ice-cold PBS and then lysed in buffer containing 20 mM Tris-HCl, pH 7.6, 0.5% Nonidet P-40, 0.25 M NaCl, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na3VO4, 0.25 µg/ml leupeptin, 1 µg/ml aprotinin. Cellular extracts were then cleared by centrifugation at 14,000 x g for 10 min at 4 °C. The protein concentration was determined using bovine serum albumin as a standard (15). For immunoprecipitations, 1 mg of cell extract was incubated with 10 µl of anti-FLAG affinity beads and rotated at 4 °C for 2 h. Samples were washed twice with 1 ml of PAN-Nonidet P-40 (10 mM Pipes, pH 7.0, 20 µg/ml aprotinin, 100 mM NaCl (PAN), and 0.5% Nonidet P-40) and once with 1 ml of PAN. Immune complexes were collected by centrifugation at 2000 x g for 2 min, and the protein-bound beads were heated at 90 °C for 5 min in Laemmli sample buffer. Samples were resolved by 8% SDS-PAGE and probed with appropriate antibodies. For Western blots, 100 µg of cellular extract was resolved by 8% SDS-PAGE, transferred to nitrocellulose, and probed with appropriate antibodies.
JNK Activity Assay—This assay was performed as previously described (16). Briefly, cell extract (40 µg of protein) was incubated with 10 µg of GST-c-Jun bound to glutathione-Sepharose and rotated for 2 h at 4 °C. After washing with PAN and PAN-Nonidet P-40 as described above, precipitated JNK was subjected to an in vitro kinase assay with [-32P]ATP and then resolved by SDS-PAGE and visualized by autoradiography.
NF-B Luciferase Assay—HEK293 EBNA cells were transiently cotransfected with 3 µg of MEKK3, K391M, or point mutants and 3 µgof NF-B luciferase reporter construct (Promega, Madison, WI). Cellular extracts were prepared 48 h post-transfection using passive lysis buffer (Promega) and cleared by centrifugation at 14,000 x g for 10 min, and 10 µg of protein was used to measure luminescence with a TD luminometer (Turner Designs, Sunnyvale, CA).
p38 MAP Kinase Assay—HEK293 EBNA cells were transiently transfected as previously described above. Cell extracts were prepared by lysis in buffer containing 20 mM Tris-HCl, pH 7.5, 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 20 mM NaF, 0.2 mM Na3VO4, 1 mM EDTA, and 1 mM EGTA. The p38 MAP kinase was immunoprecipitated as described previously (17). Briefly, 1 mg of cellular extract was incubated with polyclonal anti-p38 antibody (provided by Gary Johnson) and then rotated at 4 °C for 1 h with Protein A conjugated to Sepharose (Sigma). Protein complexes were collected by centrifugation at 2000 x g for 5 min. Samples were washed three times in lysis buffer and twice in kinase buffer containing 25 mM Hepes, pH 7.2, 25 mM -glycerophosphate, 25 mM MgCl2, 2 mM DTT, 0.1 mM Na3VO4. After the final wash, the buffer was removed from the beads, and a kinase reaction mix consisting of kinase buffer, 10 µCi of [-32P]ATP (PerkinElmer Life Sciences), and 10 µg of GST-ATF-2 was added to the beads. Samples were incubated for 20 min at 30 °C. The kinase reaction was stopped by the addition of 40 µl of 2x Laemmli sample buffer. Proteins were heated for 5 min at 90 °C and resolved by SDS-PAGE, and then the gel was dried and processed for autoradiography.
Production of Phosphoantibody Ser526—Phosphorylated and nonphosphorylated peptides, CMSGTGIRpSVTGTPY and CMSGTGIRSVTGTPY (where pS denotes phosphoserine), were synthesized corresponding to amino acids 519–532 of MEKK3 (New England Peptide, Inc.). The amino-terminal cysteine was introduced to provide a sulfhydryl moiety for conjugating to keyhole limpet hemocyanin (KLH) and a solid support. The phosphopeptide was dissolved at 1 mg/ml in PBS, and the pH was adjusted to 7.5 with 0.2 N NaOH for conjugation to KLH. The phosphopeptide (5 mg) was combined with 5 mg of KLH and incubated at 25 °C for 2 h, and the conjugated peptide was dialyzed overnight in PBS. Protein concentration was determined using the Bradford method, and the peptide-KLH conjugate was aliquoted (250 or 500 µg) for injection into New Zealand White rabbits (Covance, Denver, PA).
Both peptides were dissolved in 50 mM Tris-HCl, pH 8.5, and 5 mM EDTA before coupling to a solid support. Sulfolink coupling gel (Pierce) was brought to room temperature, and 
5 ml of gel slurry (2.5-ml column volume) was packed into a column (Econo columns; Fisher). The column was equilibrated with 6 volumes of 50 mM Tris, pH 8.0, and 5mM EDTA prior to the addition of 4 mg of peptide. Gel and peptide were incubated for 15 min at room temperature without mixing. Subsequently, the peptide and column mixture were incubated for 30 min at room temperature without mixing. The resin was washed with 3 volumes of 50 mM Tris-HCl, pH 8.5, and 5 mM EDTA, and then 50 mM cysteine (prepared in 50 mM Tris-HCl, pH 8.5, and 5 mM EDTA) was added to block nonspecific sites on the gel by applying 1 ml of cysteine for every 1 ml of gel. This reaction proceeded for 15 min with no mixing and 30 min of mixing. The resin was washed with 16 volumes of 1 M NaCl and 16 volumes of 0.05% sodium azide/PBS and then stored at 4 °C prior to use.
Phosphospecific antibody was purified via a two-step affinity column procedure. Briefly, 7 ml of rabbit antisera was applied through a 0.45-µm filter, the serum was delipidated with octanoic acid, and the IgG fraction was collected by ammonium sulfate precipitation (18). IgG was applied twice to an affinity column containing the phosphopeptide, and bound IgG was eluted with 0.1 M glycine, pH 2.7. Affinity-purified antibody was dialyzed in PBS and then applied to an affinity column containing the nonphosphopeptide, and the antibody that passed through the column (anti-phospho-Ser526 antibody) was collected, dialyzed in PBS containing 50% glycerol, and stored at 4 °C until use.
Dephosphorylation of MEKK3—HEK293 EBNA cells were transfected with FLAG-MEKK3 or an empty vector, and cell extracts were prepared in buffer containing 20 mM Tris-HCl, pH 7.6, 0.5% Nonidet P-40, 250 mM NaCl, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 0.25 µg/ml leupeptin, 1 µg/ml aprotinin, which was also supplemented with one tablet of serine and cysteine protease inhibitor (Roche Applied Science) per 10 ml of buffer. The buffer lacked serine/threonine phosphatase inhibitors, and the cell extracts were maintained on ice. Extracts were cleared of cellular debris by centrifugation at 14,000 x g for 5 min at 4 °C. Cell extracts containing MEKK3 (100 µg) were supplemented with 1.5 µl of 12 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 0.5 mM DTT, and 0.12 mg/ml bovine serum albumin. The reactions were incubated in a volume of 20 µl at 30 °C, and after 1 h, 2x Laemmli sample buffer was added to the cell extracts, and the proteins were resolved by SDS-PAGE and then immunoblotted with anti-phospho-Ser526 antibody.
Phosphorylation of MKK6—FLAG-MEKK3 was transfected into HEK293 EBNA cells and immunoprecipitated as described above. Preparation of the cDNA that encodes GST-KA-MKK6 was previously described (19), and the GST portion of the fusion protein was removed by proteolysis with Factor Xa. The kinase assay was performed as follows in buffer containing 25 mM Tris-HCl, pH 7.4, 25 mM MgCl2, 1 mM EGTA, 2 mM DTT, 25 mM -glycerophosphate, 50 µM ATP, 20 µCi of [-32P]ATP (10 Ci/mmol), and 10 µg of recombinant KA-MKK6, incubated at 30 °C for 20 min, and stopped by the addition of 40 µl of 2x Laemmli-DTT buffer. The proteins were denatured by heating for 5 min at 95 °C, resolved by 8% SDS-PAGE, and stained with Coomassie Blue, and the gel was dried prior to autoradiography.
Isolation of Proteins with GST-14-3-3—Cell extracts were prepared by lysis in buffer containing 20 mM Tris-HCl, pH 7.6, 0.5% Nonidet P-40, 0.25 M NaCl, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na3VO4, 0.25 µg/ml leupeptin, 1 mM DTT, and 1 µg/ml aprotinin. Extracts were cleared of cellular debris by centrifugation at 14,000 x g for 5 min at 4 °C. Approximately 500 µg of cellular extract was incubated with 20 µg of recombinant GST-lamin (amino acids 66–206) or GST-14-3-3 fusion proteins bound to Sepharose beads for 2 h at 4°C. Samples were then washed twice with PAN-Nonidet P-40 and once with PAN, proteins were collected at 2000 x g in a microcentrifuge for 1 min, and then the isolated proteins were incubated with cell extract containing endogenous protein phosphatases. The proteins were separated by SDS-PAGE, and after transfer to nitrocellulose, the membrane was probed with an amino-terminal antibody that recognizes total MEKK3 or phospho-Ser526, followed by goat anti-rabbit horseradish peroxidase antibody.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B Luciferase Activity—MEKK3 has previously been shown to play a critical role in the NF-B signal transduction pathway (9, 20), and therefore a NF-B luciferase assay was chosen as one method to screen point mutants in the activation loop of MEKK3. To investigate which amino acids might be essential in the regulation of NF-B-dependent transcription, each serine and threonine within the activation loop of MEKK3 was mutated to alanine by site-directed mutagenesis (Fig. 1A). Each point mutant of MEKK3 was transfected into HEK293 EBNA cells, along with a luciferase reporter gene containing the NF-B recognition sequence. After 48 h, cell extracts were prepared and assayed for luciferase activity. Expression of MEKK3 resulted in agonist-independent transcription of the NF-B luciferase reporter gene, whereas only the (S526A) and (T530S) point mutants failed to activate the reporter gene (data not shown). This result suggested that Ser526 and Thr530 are required for MEKK3-dependent regulation of the NF-B promoter. Moreover, this result strongly suggested that Ser526 and Thr530 might be phosphorylation sites within the activation loop of MEKK3.
Ser526 or Thr530 were mutated to aspartic or glutamic acid, since such mutations have been shown to produce constitutively active kinases (21, 22). Experiments were performed in which S526A, S526D, S526E, T530A, T530D, and T530E MEKK3 were transfected into HEK293 EBNA cells, along with the NF-B luciferase reporter gene. The S526A and T530A MEKK3 mutants were unable to activate transcription of the NF-B luciferase reporter gene (Fig. 1B). However, the aspartic and glutamic acid mutants of Ser526 restored 50% of wild-type MEKK3 activity, suggesting that the negative charge of the carboxyl group might mimic the negative charge of a phosphate moiety at this position. In contrast, the aspartic and glutamic acid mutants of Thr530 were unable to restore NF-B luciferase activity, indicating that a negative charge at position 530 was not tolerated within the activation loop. In addition, mutation of the active site lysine to methionine (K391M) abrogated luciferase activity, indicating that the catalytic activity of MEKK3 was required for NF-B-dependent luciferase activity. Thus, S526A and T530A MEKK3 were unable to activate transcription through the NF-B promoter, although the active site lysine was retained, demonstrating that serine 526 and threonine 530 are important for MEKK3-dependent transcription.
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B luciferase assay, we chose to investigate the role of these point mutants in activating the ERK, JNK, and p38 pathways. First, we investigated the ERK MAPK pathway. HEK293 EBNA cells were transiently transfected with MEKK3, catalytically inactive MEKK3, and the seven activation loop point mutants to determine whether the activation loop point mutants were capable of activating the ERK pathway. After 48 h, cellular extracts were prepared and resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted for phosphorylated ERK. Consistent with the luciferase assays described above, expression of (S526A) and (T530A), as well as catalytically inactive MEKK3, did not result in phosphorylation of ERK1 and -2 (Fig. 2, top). In contrast, expression of MEKK3, as well as the S511A, T516A, T520A, T522A, and the T528A point mutants resulted in ERK activation. In addition, the constitutively active mutants of Ser526, S526D and S526E, also yielded a robust phosphorylation of the ERKs, whereas the constitutively active mutants of Thr530, T530D and T530E, were not able to activate the ERKs (data not shown), which was consistent with the luciferase assay previously shown. The nitrocellulose membrane was subsequently reprobed for ERK1 and -2 as a loading control, thereby demonstrating that differences in ERK phosphorylation were due to MEKK3 activity and not ERK expression. Previous work has shown that ERK1 and -2 are phosphorylated and activated by MEK1 and -2 (23). To determine whether expression of MEKK3 affected MEK phosphorylation, the experiment described above was also probed for phosphorylated MEK1 and -2. Consistent with the ERK data, cells transfected with the S526A and T530A point mutants did not result in phosphorylation of MEK1 and -2 (Fig. 2, middle). The membrane was subsequently reprobed for MEK1 and -2 as a loading control. In addition, the membrane was also probed for MEKK3 expression by using the monoclonal antibody that recognizes the FLAG epitope, which demonstrated similar transfection efficiencies for each of the mutants. It is interesting to note that K391M, S526A, and T530A MEKK3, which are the three mutants that were unable to activate ERK and MEK phosphorylation, appeared as discreet polypeptides on the MEKK3 immunoblot, which typically indicates a lack of phosphorylation (Fig. 2, bottom, lanes c, e, and i). The reduced width of the immunoblotted protein bands probably reflects decreased phosphorylation of MEKK3 and not decreased expression (also see Fig. 5, lane g).
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-32P]ATP. Proteins were resolved by SDS-PAGE, and phosphorylated GST-ATF2 was identified by autoradiography. Consistent with previous studies, expression of MEKK3 resulted in activation of p38 MAP kinase (Fig. 3, lane b), whereas transfection of an empty vector resulted in no p38 activity (lane a). A similar phosphorylation pattern was observed from nontransfected HEK293 EBNA cells treated with 0.4 M sorbitol (lane f). However, expression of S526A and T530A MEKK3 did not activate p38 MAP kinase (lanes c and d), although the expression of MEKK3 (Fig. 3, bottom, lane b) or the mutant proteins was similar (lanes c and d). These results demonstrate that Ser526 and Thr530 are critical for MEKK3-dependent p38 MAP kinase activation.
Activation Loop Mutants S526A and T530A MEKK3 Are Unable to Activate JNK—MEKK3 has been shown to activate JNK (3, 5), and therefore we wanted to determine whether Ser526 and Thr530 were required to activate JNK. HEK293 EBNA cells were transfected with an empty vector as a control or constructs encoding various forms of MEKK3, as indicated in Fig. 3B. After 48 h, 40 µg of cell extract was incubated with GST-c-Jun to precipitate endogenous JNK. After washing the precipitated proteins with buffer, the GST-c-Jun·JNK protein complex was incubated with [-32P]ATP for 20 min, and then the proteins were resolved by SDS-PAGE. Phosphorylated GST-c-Jun was visualized by autoradiography. Expression of MEKK3 resulted in a robust activation of JNK (Fig. 3, lane b), whereas expression of an empty vector as a control (lane a), catalytically inactive MEKK3 (lane c), or the activation loop point mutants (lanes d and e) resulted in very little JNK activity. In summary, we demonstrate that the catalytic activity of MEKK3 is required to activate the ERK, p38, and JNK MAP kinases. In addition, mutation of Ser526 and Thr530 to alanine, while retaining the active site lysine at position 391, also produced catalytically inactive mutants of MEKK3. Thus, Ser526 and Thr530 are critical amino acids within the activation loop of MEKK3.
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90 kDa (lanes a–e). Importantly, the phospho-Ser526 antibody showed no immunoreactivity toward cell extracts expressing S526A MEKK3 (lane d), indicating that the antibody was specific for phospho-Ser526. In addition, catalytically inactive MEKK3 was not detected by the phosphoSer526 antibody (lane c), strongly suggesting that phosphorylation of Ser526 occurs via autophosphorylation of MEKK3 and not by an upstream kinase. If phosphorylation of MEKK3 occurred via an upstream kinase that is endogenously expressed in HEK293 cells, one would expect phosphorylation of catalytically inactive MEKK3 at Ser526. It should be emphasized that the catalytically inactive mutant of MEKK3 can be phosphorylated at Ser166, which demonstrates that this mutant form of MEKK3 retains its structure for phosphorylation by another kinase (12), and presumably the structurally features surrounding Ser526 in the catalytically inactive mutant resemble wild-type MEKK3. This result demonstrates that the catalytically inactive mutant of MEKK3 retains the structural features needed for phosphorylation by another kinase and indicates that phosphorylation of Ser526 occurs by autophosphorylation. Finally, it is interesting to note that T530A MEKK3 was recognized by the anti-phospho-Ser526 antibody (lane e), albeit to a lesser extent than wild type MEKK3. This result suggests that Thr530 is not essential for phosphorylation of Ser526, although Thr530 is required for signaling to downstream substrates. Reprobing the immunoblot with a monoclonal antibody that recognizes the FLAG epitope of transfected MEKK3 indicated that similar levels of the MEKK3 proteins were expressed in the cells (Fig. 4A, bottom).
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Dephosphorylation of Ser526—By developing an antibody that recognized Ser526 when phosphorylated, we could determine whether Ser526 was sensitive to dephosphorylation. Since expression of MEKK3 in HEK293 EBNA cells resulted in phosphorylation of Ser526, we postulated that protein phosphatase(s) expressed in this cell line would also dephosphorylate the site. To test this hypothesis, HEK293 EBNA cells were transfected with a cDNA that encodes MEKK3 (Fig. 5, lanes f–j) or an empty vector (lanes a–e), and after 48 h, cell extracts were prepared at 4 °C in buffer lacking serine/threonine phosphatase inhibitors. After cell debris was removed by centrifugation, 100 µg of cell extract in 20 µl was supplemented with 1.5 µl of 12mM Tris, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 0.5 mM DTT, and 0.12 mg/ml bovine serum albumin. Then the cell extracts were incubated at 30 °C for 1 h. Where indicated, phosphatase reactions also included 10 mM NaF or 100 nM okadaic acid. As we have previously shown, expression of MEKK3 in HEK293 EBNA cells resulted in phosphorylation of MEKK3 at Ser526 (Fig. 5, top, lane f). In this experiment, we demonstrate that incubation of MEKK3 in the HEK293 EBNA cell extract resulted in complete dephosphorylation of MEKK3 (Fig. 5, top, lane g). Immunoblotting the same nitrocellulose membrane with antibody that recognizes the amino terminus of MEKK3 demonstrated that the lack of immunoreactivity in lane g was not due to degradation of MEKK3. In fact, the loss of phosphate from MEKK3 is reflected by the increased mobility and decreased molecular mass of MEKK3 by SDS-PAGE (Fig. 5, bottom, compare lane g with lane f). Moreover, serine/threonine phosphatase inhibitors such as NaF and okadaic acid were included in the phosphatase reactions and were highly effective at inhibiting the phosphatase activity in the HEK293 EBNA cell extracts (Fig. 5, top, lanes h–j). In summary, these data demonstrate that Ser526 of MEKK3, when incubated in cell extracts, is susceptible to dephosphorylation and that the phosphatase(s) responsible for this activity is sensitive to NaF and okadaic acid.
Dephosphorylation of Ser526 by PP2A Isoforms—Since we had established dephosphorylation of Ser526 when MEKK3 was incubated in cell extracts, we tested whether the protein serine/threonine phosphatase, PP2A, could dephosphorylate Ser526. To obtain two different PP2A holoenzymes, HEK293 cells were transfected with either the FLAG-tagged 
or 
isoform of the regulatory B subunit of PP2A. The A subunit is a scaffolding protein, whereas the C subunit is the catalytic protein of PP2A. The PP2A holoenzymes, consisting of either ABC or ABC subunits, were isolated by immunoprecipitation using the monoclonal antibody that recognizes the FLAG epitope. The PP2A holoenzymes were eluted from the Sepharose beads using the FLAG peptide at a concentration of 0.1 mg/ml as previously described (24). In a separate transfection, HEK293 EBNA cells were transfected with FLAG-tagged MEKK3, and after 48 h, cell extracts were prepared, and tagged MEKK3 was immunoprecipitated using the FLAG monoclonal antibody. Immunoprecipitated FLAG MEKK3 was incubated with 9 ng of PP2A holoenzymes, ABC (Fig. 6, lanes b and c) orABC (Fig. 6, lanes d and e) for 30 or 60 min at 30 °C. Within 30 min, phospho-Ser526 was completely dephosphorylated by either PP2A holoenzyme. These results suggest that the protein phosphatase that dephosphorylates Ser526 from HEK293 EBNA cell extracts is probably an isoform of PP2A.
Dephosphorylation of Ser526 Affects MEKK3 Catalytic Activity toward MKK6—Since MEKK3 is a MAP3K that functions upstream of the p38 MAP kinase and we had shown the phospho-Ser526 was required for MEKK3-dependent activation of p38 (Fig. 3A), we tested whether phosphorylation of Ser526 was required for MEKK3 to directly phosphorylate a substrate like MKK6. The cDNA that encodes FLAG-MEKK3 was transfected into HEK293 EBNA cells, and after 48 h, Sepharose beads conjugated to the monoclonal antibody that recognizes the FLAG epitope were used for immunoprecipitation. Precipitated proteins were washed twice with 1 ml of PAN Nonidet P-40 buffer, once with 1 ml of PAN buffer, and aliquoted into two tubes. After centrifugation to collect the protein-bound beads, 100 µg of nontransfected HEK293 EBNA cell extract that was prepared with 10 mM NaF and 100 nM okadaic acid (Fig. 7, A (lanes a and b), B (lane b), and C (lane a)) or without phosphatase inhibitors (Fig. 7, A (lanes c and d), B (lane b), and C (lane b)) was added to each tube and incubated for 1 h at 30 °C.After the dephosphorylation reaction, 20% of the sample was removed to assess dephosphorylation of FLAG-MEKK3 by immunoblotting with antibody that recognizes phosppho-Ser526 (Fig. 7B), whereas the remaining sample was washed twice with PAN buffer and aliquoted again into two tubes to perform a kinase assay. An in vitro kinase assay was performed with KA-MKK6 as substrate.
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Association of MEKK3 with 14-3-3 Prevents Dephosphorylation of Ser526—Serine and threonine phosphorylation of proteins is frequently accompanied by the recruitment of 14-3-3 proteins (25), which affects protein/protein interactions, subcellular localization, and activity (reviewed in Refs. 26 and 27). Since we had previously demonstrated an association between MEKK3 and 14-3-3 (28), we wanted to determine whether the association between MEKK3 and 14-3-3 proteins required phosphorylation of Ser526. HEK293 EBNA cells were transfected with FLAG MEKK3 or point mutants, and after 48 h, cell extracts were prepared, and the MEKK3 proteins were immunoprecipitated using the FLAG epitope. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibody that recognizes the phospho-Ser526 epitope. As shown previously in Fig. 4, MEKK3 was phosphorylated on Ser526, and the T530A point was also weakly phosphorylated on Ser526. In contrast, the S526A point mutant and the kinase-inactive mutant K391M were not phosphorylated on Ser526 (Fig. 8, top). The nitrocellulose membrane was also probed for the 14-3-3 protein, and the co-precipitation of 14-3-3 protein was evident with wild-type MEKK3 and weakly with the T530A mutant, whereas mutants that were not phosphorylated on Ser526 were also unable to associate with 14-3-3 protein. From this experiment, it is not known which isoform of 14-3-3 interacts with MEKK3, since the 14-3-3 antibody recognizes amino acids 3–21 of the human isoform of 14-3-3, and this sequence is highly conserved within the 14-3-3 family of proteins (, , , , , , and ).
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isoform of 14-3-3 (28), we wanted to determine whether the association between MEKK3 and 14-3-3 would prevent dephosphorylation of Ser526. HEK293 EBNA cells were transfected with FLAG MEKK3, and cell extracts were prepared in the absence of okadaic acid and sodium fluoride. Then the cell extracts were incubated at 4 °C with GST-14-3-3 or GST-lamin that was expressed and purified from bacteria, as previously described (28). After 1 h, the GST fusion proteins and associated proteins were collected by centrifugation and washed twice with 1 ml of PAN-Nonidet P-40 and PAN buffers to remove nonspecific proteins. Precipitated proteins were resolved by SDS-PAGE and immunoblotted using the anti-phospho-Ser526 antibody. 14-3-3 precipitated FLAG-MEKK3 while phosphorylated at Ser526 (Fig. 9, top, lane a), whereas the lamin fusion protein was unable to precipitate FLAG-MEKK3 (lane g), as we have previously reported (28).
Since we were able to demonstrate dephosphorylation of Ser526 as shown in Fig. 5, an experiment was designed to determine whether the association between MEKK3 and 14-3-3 prevented dephosphorylation of Ser526. FLAG-MEKK3 was precipitated from HEK293 EBNA cell extracts with GST-14-3-3, as described above, and then incubated with 100 µg of HEK293 EBNA cell extract from nontransfected cells and incubated at 30 °C for up to 3 h. The phosphatase reactions were terminated by the addition of Laemmli sample buffer, and the proteins were resolved by SDS-PAGE and immunoblotted with anti-phospho-Ser526 MEKK3 antibody. The association of FLAG-MEKK3 with 14-3-3 prevented the dephosphorylation of Ser526 (Fig. 9, top, lanes b–f). In contrast, incubation of cell extracts containing FLAG-MEKK3, under the same conditions that promote phosphatase activity, resulted in complete dephosphorylation of Ser526 after 30 min (lane j), whereas the amount of FLAG-MEKK3 that was used in each experimental condition was similar, as determined by immunoblotting with antibody that recognizes the amino terminus of MEKK3 (Fig. 9, bottom, compare lanes a–f with lanes i–k). In summary, the association between FLAG-MEKK3 and 14-3-3 inhibits dephosphorylation of Ser526. Thus, given the importance of phospho-Ser526 in regulating MEKK3 activity, it appears that the interaction between MEKK3 and 14-3-3 is critical for maintaining a catalytically active form of MEKK3.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In this study, we demonstrate that Ser526 is the only phosphorylation site within the activation loop of MEKK3 and that phosphorylation occurs in response to exogenous stimuli like osmotic stress. In addition, whereas mutation of other putative phosphorylation sites did not significantly alter MEKK3 activity, mutation of Thr530 to a conserved alanine or to the phosphomimetic amino acids, aspartate or glutamate, was not tolerated, suggesting that Thr530 might play an important role in maintaining the structural integrity of the activation loop. Although the crystal structure of the catalytic domain of MEKK3 has not been resolved, the crystal structure of the catalytic subunit of PKA provides insights as to the orientation of the activation loop of MEKK3. Two critical amino acids between subdomains VII and VIII of the catalytic subunit of PKA, Thr197 and Thr201, are localized at positions equivalent to those of Ser526 and Thr530 of MEKK3, respectively. In PKA, Thr197 is phosphorylated and critical for activity, whereas Thr201 is also critical for activity but not a phosphorylation site (32). In this study, we have demonstrated that Ser526 of MEKK3 is phosphorylated and critical for catalytic activity, like Thr197 of PKA. We have also shown that Thr530, which corresponds to Thr201 of PKA, is required for MEKK3 activity and not likely to be a phosphorylation site. This result suggests that Thr530 is critical to maintain the structural integrity of the activation loop of MEKK3. In the crystal structure of PKA that is bound to substrate peptide, Thr201 of the PKA catalytic subunit interacts with Asp166 but only in a substrate-dependent manner (32). Given the similarities between the PKA and MEKK3 activation loops, it is quite possible that Thr530 of MEKK3 interacts with Asp489, which corresponds to Asp166 of PKA. The fact that alanine or negatively charged amino acids are not tolerated at position 530 supports the notion that the hydroxyl moiety of Thr530 interacts with a negatively charged amino acid, like Asp489. The crystal structure of the MEKK3 catalytic domain while bound to substrate peptide should confirm this prediction.
The development of an antibody that recognizes phospho-Ser526 provides compelling evidence that Ser526 is a phosphorylation site within the activation loop of MEKK3. Further evidence that a negative charge at Ser526 is critical for MEKK3 activity comes from experiments using the S526D and S526E mutants of MEKK3, which activated the ERK MAP kinases, MEK, and NF-B luciferase activity. In contrast, negatively charged amino acids at Thr530 produced inactive mutants of MEKK3. Moreover, attempts to detect phospho-Thr530 of MEKK3 with a phosphospecific antibody were unsuccessful (data not shown), which supports the conclusion that Thr530 is not a phosphorylation site. However, although Thr530 is not a phosphorylation site, Thr530 is required for the phosphoryl transfer reaction catalyzed by MEKK3.
The catalytically inactive mutant of MEKK3, which has the active site lysine mutated to methionine (K391M), was not phosphorylated on Ser526. Based on this observation, our results suggest that phosphorylation of Ser526 occurs by an autophosphorylation mechanism. If phosphorylation of Ser526 had occurred in the K391M MEKK3 mutant, such a result would have indicated that an upstream kinase in the signaling cascade, such as a mitogen-activated protein kinase kinase kinase kinase, phosphorylates Ser526. But this result was never observed in these studies. It is interesting to note that although Thr530 is required for MEKK3-dependent MAP kinase activity, autophosphorylation of Ser526 does not require Thr530, albeit efficient autophosphorylation of Ser526 may require Thr530. Nonetheless, our results suggest that the autophosphorylation of MEKK3 may occur via a different mechanism from phosphorylation of downstream kinases, such as the MAP2K family. Since the MKKs require dual phosphorylation for efficient catalytic activity, it is possible that Thr530 is necessary to orient the activation loop of MEKK3 for dual phosphorylation of MKKs, whereas Thr530 is not necessary for phosphorylation of Ser526. Additional mechanistic studies will be needed to delineate the difference between those phosphorylation events.
Dephosphorylation of Ser526 was readily accomplished by using purified PP2A holoenzymes. Moreover, the presence of PP2A inhibitors such as okadaic acid or sodium fluoride in cell extracts containing transfected MEKK3 prevented dephosphorylation of Ser526, and the absence of phosphatase inhibitors resulted in dephosphorylation of Ser526. The reversible nature of Ser526 phosphorylation further supports the fact that a post-translational modification of Ser526 is physiologically relevant for MEKK3 activity, especially under conditions of osmotic stress. The study described herein also demonstrates that phospho-Ser526 is also required for efficient phosphorylation of downstream substrates, such as MKK6. Importantly, dephosphorylation of Ser526 is prevented by the interaction with 14-3-3 protein, which is consistent with the previously defined role of 14-3-3 proteins binding to phosphorylated serine or threonine (33). However, phospho-Ser526 is not localized within a consensus 14-3-3 binding site as described by Yaffe et al. (34). Although we know that 14-3-3 binding to MEKK3 prevents dephosphorylation of MEKK3, we do not know whether phospho-Ser526 contributes directly to the binding with 14-3-3 protein. Our previous study using the amino terminus of MEKK3 as bait in the yeast two-hybrid system suggests that Ser526 is not directly involved in 14-3-3 binding, since we have shown that the amino terminus is sufficient to interact with 14-3-3 proteins (28). Thus, perhaps other phosphorylation sites on MEKK3, such as Ser166 and/or Ser337, are needed for binding with 14-3-3 (12) and the tertiary structure of MEKK3, when bound to 14-3-3, does not allow phosphatases to access phosphorylated Ser526.
Future studies must now be directed toward understanding the mechanism by which MEKK3 autophosphorylation occurs and, conversely, how protein phosphatases like PP2A dephosphorylate Ser526.It is clear that the interaction between MEKK3 and 14-3-3 proteins helps maintain phosphorylation of Ser526, but what causes the dissociation of 14-3-3 proteins from phosphorylated MEKK3 is a question whose answer will probably allow us to better understand how MEKK3 functions in the cell. In summary, Ser526 is an activation loop phosphorylation site that is critical for MEKK3 activity, much as phosphorylation of the activation loop of Thr197 of the catalytic subunit of PKA functions as a molecular switch for enzymatic activity (35). Moreover, Ser526 provides a molecular target to readily assess MEKK3 activity under different physiological conditions.
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FOOTNOTES |
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1 To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, 1703 E. Mabel St., Tucson, AZ 85721. Tel.: 520-626-4374; Fax: 520-626-2466; E-mail: vaillancourt{at}pharmacy.arizona.edu.
2 The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEKK3, MAPK/ERK kinase kinase 3; JNK, c-Jun NH2-terminal kinase; MAP2K, MAPK kinase; MAP3K, MAPK kinase kinase; PP2A, protein phosphatase 2A; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; DTT, dithiothreitol; KLH, keyhole limpet hemocyanin; PBS, phosphate-buffered saline; GST, glutathione S-transferase; PKA, protein kinase A; EBNA, Epstein-Barr virus nuclear antigen.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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