Phosphorylation of Thr-516 and Ser-520 in the Kinase Activation Loop of MEKK3 Is Required for Lysophosphatidic Acid-mediated Optimal IκB Kinase β (IKKβ)/Nuclear Factor-κB (NF-κB) Activation*

MEKK3 serves as a critical intermediate signaling molecule in lysophosphatidic acid-mediated nuclear factor-κB (NF-κB) activation. However, the precise regulation for MEKK3 activation at the molecular level is still not fully understood. Here we report the identification of two regulatory phosphorylation sites at Thr-516 and Ser-520 within the kinase activation loop that is essential for MEKK3-mediated IκB kinase β (IKKβ)/NF-κB activation. Substitution of these two residues with alanine abolished the ability of MEKK3 to activate IKKβ/NF-κB, whereas replacement with acidic residues rendered MEKK3 constitutively active. Furthermore, substitution of these two residues with alanine abolished the ability of MEKK3 to mediate lysophosphatidic acid-induced optimal IKKβ/NF-κB activation.

One of the critical steps for kinase activation is phosphorylation of the specific serine or threonine residues within the activation loop of the protein kinase located between kinase subdomains VII and VIII (23). In the case of MEKK3, several studies have been carried out to identify the phosphorylation sites and the effect of phosphorylation on MEKK3 activity. For example, phosphorylation of Ser-166 and Ser-337 has been shown to be promoted by tumor necrosis factor ␣ but not required for MEKK3 activity (24), and phosphorylation of Ser-526 is required for MEKK3-mediated MAPK and NF-B activation (25,26). However, the molecular mechanism of MEKK3-mediated IKK␤/NF-B activation remains to be clearly defined.
In this study, we further examine the potential phosphorylation sites in the kinase activation loop of MEKK3 that is required for MEKK3-mediated NF-B activation. Using mutational analysis and reporter assays, we identified Thr-516 and Ser-520 within kinase subdomains VII and VIII as two additional regulatory phosphorylation sites required for MEKK3-mediated IKK␤/NF-B activation. We demonstrate that both Thr-516 and Ser-520 on MEKK3 are phosphorylated by using specific antibodies that recognize the phosphorylation of these two sites. Phosphorylation of these two residues on MEKK3 is required for LPA-induced optimal IKK␤/NF-B activation as well as interleukin-6 (IL-6) production.
Luciferase Reporter Gene Assay-Luciferase reporter gene assay was performed using a Dual-Luciferase reporter assay system (Promega, Madison, WI) and a Monolight 3010 luminometer (Pharmingen) as described previously (28). Briefly, targeted cells were transiently cotransfected with specific vectors and an NF-B-dependent firefly luciferase reporter construct as well as a Renilla luciferase control construct. Cellular extracts were prepared 36 h post-transfection, and the luciferase activities were determined. Relative NF-B luciferase activity was normalized to Renilla luciferase activity. Changes in luciferase activity with respect to control were calculated. Each experiment was conducted in triplicate.
Preparation of Nuclear and Cytosolic Fractions-Nuclear and cytosolic extracts were made as described (27). In brief, cells were harvested in ice-cold phosphate-buffered saline (pH 7.4) and pelleted by centrifugation at 500 ϫ g for 3 min and then lysed for 30 min on ice in buffer A (10 mM HEPES buffer (pH 7.9) containing 0.1 mM EDTA, 10 mM KCl, 0.4% (v/v) IGEPAL, 0.5 mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonyl fluoride). Lysates were centrifuged at 15,000 ϫ g for 10 min. The resulting supernatants constituted cytosolic fractions. The pellets were washed three times with buffer A and resuspended in buffer B (20 mM HEPES buffer (pH 7.9) containing 400 mM NaCl, 1 mM EDTA, 0.5 mM DTT, and 1 mM phenylmethylsulfonyl fluoride), incubated for 30 min on ice, and centrifuged at 15,000 ϫ g for 10 min. The supernatants were used as nuclear extracts.
Immunoblotting and Immunoprecipitation-Cells were harvested in ice-cold phosphate-buffered saline (pH 7.4) and spun down. The pellet was dissolved in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% IGEPAL, 0.25% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM benzamidine, 20 mM disodium p-nitrophenyl phosphate, 0.1 mM sodium orthovanadate, 10 mM sodium fluoride, and phosphatase inhibitor mixture A and B (Sigma)). The cell lysates were either subjected directly to 10% SDS-PAGE for immunoblotting analysis or immunoprecipitated 3 h with the indicated antibodies. Immune complexes were recovered with Protein A-agarose (Santa Cruz Biotechnology) for 3 h and then washed three times with wash buffer containing 20 mM HEPES (pH 7.4), 50 mM NaCl, 2.5 mM MgCl 2 , 0.1 mM EDTA, and 0.05% Triton X-100. For immunoblotting, the immunoprecipitates or whole cell lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). The membranes were immunoblotted with various antibodies, and the bound antibodies were visualized with horseradish peroxidase-conjugated antibodies against rabbit or mouse IgG using the ECL-Plus Western blotting system according to the manufacturer's instructions.
In Vitro Dephosphorylation Assay of Phospho-MEKK3 by PP1 or -PPase-HEK-293T cells were transfected with a HA-MEKK3 expression plasmid or an empty vector. Thirty-six hours after transfection, cell extracts were prepared in buffer containing 50 mM Tris-HCl (pH 7.4) 150 mM NaCl, 1 mM EDTA, 1% IGEPAL, 0.25% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT, 10 g/ml aprotinin, 10 g/ml leupeptin, and 1 mM benzamidine. The HA-tagged MEKK3 protein was immunoprecipitated 3 h with anti-HA antibody-conjugated Protein A-agarose and washed three times with lysis buffer and twice with phosphatase reaction buffer (50 mM HEPES, 100 mM NaCl, 2 mM DTT, 0.1 mM EGTA, 0.025% Tween 20, 1 mM MnCl 2 (pH 7.5)). One unit of PP1 or -PPase with phosphatase reaction buffer was then added into the tubes containing the precipitated MEKK3 protein incubated for 25 min at 30°C. Laemmli sample buffer was added to the reaction and boiled at 95°C for 5 min, and the proteins were resolved by SDS-PAGE and then immunoblotted with antiphospho-MEKK3 antibodies.
Electrophoretic Mobility Shift Assay-NF-B oligonucleotide probes were labeled with [␥-32 P]ATP. MEF cells (1 ϫ 10 6 ) were starved for 12 h and stimulated for the indicated time points. Nuclear extracts isolated from these cells were then incubated with 32 P-labeled probes in 10 mM HEPES (pH 7.9), 40 mM NaCl, 1 mM EDTA, 4% glycerol, 3 g of poly(dI⅐dC), and 0.5 mM DTT for 15 min at room temperature. The samples were then resolved in a nondenaturing polyacrylamide gel and exposed to x-ray film at Ϫ80°C.

JOURNAL OF BIOLOGICAL CHEMISTRY 7913
Cell Proliferation Assay-Empty vector-, MEKK3-WT-, and MEKK3-AA-reconstituted MEF cells were seeded on 96-well plates with 1 ϫ 10 4 cells/well. Twenty-four hours later, cells were treated with or without LPA (30 M) or PMA (40 ng/ml) plus Iono (100 ng/ml). Cell numbers in each well were counted after treatment for 24, 48, and 72 h. Each experiment was conducted in triplicate.

RESULTS
Thr-516 and Ser-520 within the Activation Loop of Human MEKK3 Are Two Potential Phosphorylation Sites Essential for MEKK3-mediated NF-B Activation-Phosphorylation of serine and/or threonine residue(s) within kinase subdomains VII and VIII is essential for the activation of many kinases involved in the regulation of NF-B activation such as IKK (23). We aligned amino acid sequences of the MEKK3 kinase activation loop from various species and indicated five conserved Ser/Thr sites (Ser-511, Thr-516, Ser-520, Thr-522, and Ser-526) (Fig. 1A).
Phosphorylation of Ser-526 has been identified to be essential for MEKK3-mediated MAPK and NF-B activation (25,26). To identify other potential phosphorylation sites on MEKK3 that are essential for its NF-B activation, an NF-B-dependent luciferase reporter assay was chosen to screen the effect of point mutants of serine/threonine residues within the activation loop of MEKK3 on its NF-B activation. We systematically mutated five serine and threonine residues to alanine within the kinase activation loop between kinase subdomains VII and VIII of human MEKK3 to examine whether these mutations would affect MEKK3-induced NF-B activation in a reporter assay (Fig. 1B). The expression plasmid encoding wild-type MEKK3, the active site lysine-to-arginine mutant (K391R), or each point mutant of MEKK3 was transfected into HEK-293T cells, along with plasmids containing an NF-B-dependent firefly and a control pCMV-Renilla luciferase reporter gene. As shown in Fig. 1B, three MEKK3 mutations (T516A, S520A, and S526A) resulted in the inhibition of MEKK3-induced NF-B luciferase reporter gene expression in the cells. These results suggest that Thr-516 and Ser-520, in addition to Ser-526, are also potential phosphorylation sites essential for MEKK3-mediated NF-B activation.
Because Ser-526 has been reported to be essential for MEKK3-mediated NF-B activation, we hypothesized that Thr-516 and Ser-520 are additional phosphorylation sites con- tributing to MEKK3-mediated NF-B activation. To test this hypothesis, we made substitutions of Thr-516 and Ser-520 with single or double acidic residues (MEKK3-T516E and MEKK3-S520D) to mimic the phosphorylation of these two residues. As shown in Fig. 1C, MEKK3-T516E/S520D double mutants induced NF-B activation in the reporter assay at a much higher level compared with wild-type MEKK3 or the T516E or S520D single mutant, whereas the T516E/S520A, T516A/S520D, and T516A/T520A double mutants failed to activate the NF-B reporter gene. These results strongly suggest that the MEKK3-T516E/S520D mutant is constitutively active in the assay and that phosphorylation of both Thr-516 and Ser-520 is required for the MEKK3-mediated NF-B activation.
Successful Generation of Anti-phospho-MEKK3 (pThr-516/ pSer-520) Antibodies, and LPA Induces MEKK3 Phosphorylation at Thr-516 and Ser-520-To examine directly whether Thr-516 and Ser-520 of human MEKK3 are phosphorylated during its activation, rabbit antibodies specifically recognizing Thr-516-and Ser-520-phosphorylated human MEKK3 were generated by immunizing rabbits with a synthetic phosphopeptide (keyhole limpet hemocyanin-coupled) corresponding to residues surrounding Thr-516 and Ser-520 of human MEKK3. Antibodies were purified by Protein A-agarose and peptide affinity chromatography. As shown in Fig. 2A, our antibodies specific for phospho-MEKK3 (pThr-516/pSer-520) strongly recognized wild-type MEKK3 but not T516A/S520A and T516E/S520D double mutant proteins. Furthermore, our antibodies also weakly recognized MEKK3-T516A and MEKK3-S520A single mutant proteins. To further validate the specificity of our antibodies, a mammalian expression vector encoding wild-type HA-tagged MEKK3 was transfected into HEK-293T cells. Overexpressed MEKK3 proteins were immunoprecipitated from cell lysates and treated with PP1 or -PPase and resolved by 10% SDS-PAGE. As shown in Fig. 2B, our antibodies recognized only the nontreated but not the phosphatasetreated wild-type MEKK3, although the same amount of MEKK3 protein was loaded. These results demonstrate that our antibodies specifically detect MEKK3 only when phosphorylated at Thr-516 and/or Ser-520.
Phosphorylation of Thr-516 and Ser-520 Is Required for MEKK3mediated IKK/NF-B Activation-Phosphorylation of IKK␤ Ser-177 and Ser-181 in the kinase activation loop is essential for IKK activation (23). Overexpression of MEKK3 results in IKK activation (27,29). To test whether phosphorylation of both Thr-516 and Ser-520 sites is required for MEKK3-mediated IKK phosphorylation at Ser-177 and Ser-181, we transfected the full-length wild-type and mutant MEKK3 expression plasmids with HA-tagged IKK␤ into Mekk3 Ϫ/Ϫ MEF cells. The HA-tagged IKK␤ proteins were immunoprecipitated from the cell lysates with anti-HA antibodies and immunoblotted with antibodies specific for phospho-IKK␣/␤. As shown in Fig. 3A, overexpression of only MEKK3-WT and the MEKK3-T516E/S520D mutant resulted in IKK␤ phosphorylation at Ser-177 and Ser-181, whereas the MEKK3-T516E/ S520D mutant induced a higher level of IKK␤ phosphorylation compared with MEKK3-WT. Other mutants were not able to induce the visible IKK␤ phosphorylation at all. Consistent with these results, luciferase analysis with an NF-Bresponsive reporter showed that only wild-type and T516E/ S520D mutant MEKK3 significantly induced the activity of NF-B in Mekk3 Ϫ/Ϫ MEF cells (Fig. 3B). Previous studies demonstrate that MEKK3 Ser-526 is essential for MEKK3-mediated NF-B activation (25,26). Interestingly, we found the MEKK3-T516E/S520D/S526A mutant failed to induce NF-B reporter activity compared with MEKK3-WT and the MEKK3-T516E/S520D mutant (Fig. 3C). This result indicates that MEKK3 phosphorylation at Thr-516/ Ser-520/Ser-526 is essential for MEKK3-mediated NF-B activation.
Taken together, our results suggest that phosphorylation of Thr-516 and Ser-520 sites is required for MEKK3-mediated IKK␤/NF-B activation. Once overexpressed, the MEKK3-T516E/S520D mutant acts as a constitutively active mutant to induce IKK␤/NF-B activation.
Phosphorylation of Thr-516 and Ser-520 on MEKK3 Is Required for LPA-and PKC-induced IKK/NF-B Activation-MEKK3 has been shown to be involved in the regulation of IKK/NF-B activation (27,29,30). MEKK3 is required for LPA-induced NF-B activation (22). In this study, mutation of Thr-516 and Ser-520 to alanine abolished MEKK3-mediated IKK/NF-B activation. Therefore, we hypothesized that phosphorylation of Thr-516 and Ser-520 on MEKK3 plays an important role in LPAmediated IKK/NF-B activation. To test this hypothesis, HA-MEKK3-WT and HA-MEKK3-AA mutant expression vectors, as well as an empty vector, were stably introduced back into Mekk3 Ϫ/Ϫ MEF cells by the retroviral infection system. Stable MEF cell lines expressing MEKK3-WT (HA-MEKK3-WT), T516A/S520A (HA-MEKK3-AA) mutants, and empty control vector were established. MEKK3 expression was verified by immunoblotting (Fig. 4A).
To further characterize the physiological role of the phosphorylation of Thr-516 and Ser-520 on MEKK3 in LPA-and PKC-mediated IKK/NF-B activation, Mekk3 Ϫ/Ϫ MEF cell lines reconstituted with control vector, HA-MEKK3-WT, or HA-MEKK3-AA were treated with LPA or PMA/ Iono (PKC agonists) at different time points and subsequently lysed. The cell lysates were immunoblotted with the indicated antibodies to examine LPA-and PKC-induced IKK and IB␣ phosphorylation. As shown in Fig. 4, A and B, LPA and PMA/Iono induced the phosphorylation of IKK␤ and IB␣ at a much higher level in the MEKK3-WT-reconstituted MEF cells compared with the MEKK3-AA mutant-and empty vector-reconstituted cells. In addition, LPA and PMA/Iono induced a much higher level of NF-B nuclear translocation and activation in the MEKK3-WTreconstituted cells compared with the MEKK3-AA-reconstituted cells (Fig.  4, C-F). These results demonstrate that phosphorylation of Thr-516 and Ser-520 on MEKK3 is required for LPA-and PKC-induced IKK␤ phosphorylation, IB␣ phosphorylation, and NF-B activation.
Phosphorylation of Thr-516 and Ser-520 on MEKK3 Is Required for LPA-and PMA/Iono-induced IL-6 Production and Cell Proliferation-MEKK3 has been shown to be required for LPA-and PKC-mediated NF-B-induced IL-6 production in MEF cells (17,19,20). To determine the role of phosphorylation of Thr-516 and Ser-520 on MEKK3 in LPA-induced IL-6 production, the vector control-and HA-MEKK3-WT-or HA-MEKK3-AA-reconstituted Mekk3 Ϫ/Ϫ MEF cell lines were treated with or without LPA or PMA/Iono and then analyzed for IL-6 production in the cells by ELISA (Fig. 5, A and B). In this assay, both LPA and PMA/Iono induced a much higher level of IL-6 production in the MEKK3-WT-reconstituted MEF cells compared with the control and MEKK3-AA-reconstituted cells (Fig. 5, A and B). In addition, we found that LPA-and PMA/ Iono-induced cell proliferations were impaired in the vector control-and MEKK3-AA-reconstituted cells compared with the MEKK3-WT-reconstituted MEF cells (Fig. 5, C and D). These results demonstrate that phosphorylation of Thr-516 and Ser-520 on MEKK3 is essential for the LPA-and PKCinduced physiological effect in MEF cells.

DISCUSSION
Our previous study demonstrates that MEKK3 is required for the LPA-induced IKK␤/NF-B signal transduction pathway (22). However, the molecular regulation of MEKK3-mediated NF-B activation in LPA signaling remains to be better defined. Here we have presented evidence that both Thr-516 and Ser-520 within the MEKK3 kinase activation loop are two essential regulatory phosphorylation sites for MEKK3-mediated IKK␤/NF-B activation. This phosphorylation of MEKK3 is confirmed by antibodies that recognize pThr-516 and pSer-520 within the activation loop. Further evidence to support the importance of a negative charge at Thr-516 and Ser-520 on MEKK3 comes from experiments using the T516E and S520D mutants of MEKK3, which act as constitutively active mutants to induce IKK␤/NF-B activation. We propose that phosphorylation of these two residues within the kinase activation loop is a general mechanism for MEKK3-mediated NF-B activation.
Our initial luciferase reporter analysis confirmed that Ser-526 is a key regulatory residue whose phosphorylation is essential for MEKK3 to activate both NF-B and MAPK pathways (25,26). Interestingly, we found that overexpression of the T516E/S520D/S526A mutant failed to induce NF-B activation in a reporter assay. This result suggests that phosphorylation of these three sites is essential for MEKK3-mediated IKK␤/NF-B activation.
Previously, PKC␦ and PKC have been suggested to be involved in LPA-induced NF-B activation (31). Further studies are needed to determine the mechanism of PKC-mediated MEKK3 activation in LPA signaling.
In conclusion, our data provide evidence that phosphorylation of MEKK3 at Thr-516 and Ser-520 is required for LPA-and PKC-induced IKK␤/NF-B activation in MEF cells. In view of the data presented here and in previous reports, we propose a working model (Fig. 6) in which phosphorylation of MEKK3 at Thr-516, Ser-520, and Ser-526 induced by LPA-mediated PKC activation is essential for MEKK3-mediated IKK␤/NF-B activation in the cells.