Phosphorylation of MEKK3 at Threonine 294 Promotes 14-3-3 Association to Inhibit Nuclear Factor κB Activation

The protein kinase MEKK3 is essential for tumor necrosis factor α (TNFα)- and lipopolysaccharide-induced activation of nuclear factor κB, although the mechanism by which TNF receptor 1 and Toll-like receptors regulate MEKK3 is largely unknown. In this study we have identified MEKK3 Thr294 as a novel site of phosphorylation that regulates MEKK3 binding with 14-3-3. Phosphorylation of MEKK3 at Thr294 was observed for both endogenous and ectopically expressed MEKK3. Mutation of Thr294 to alanine abolished 14-3-3-MEKK3 association and incubation with phosphorylated peptides mimicking Thr(P)294 competed for 14-3-3 binding. Mutation of Thr294 did not alter Ser526 phosphorylation within the activation loop. However, expression of T294A MEKK3 elevated TNFα-stimulated NF-κB transcriptional activity, suggesting that Thr294 phosphorylation and 14-3-3 binding negatively regulate MEKK3. Stimulation with TNFα or lipopolysaccharide caused a rapid decrease in Thr294 phosphorylation of endogenous MEKK3 and subsequent loss of 14-3-3 association. Thus, this study identifies a potentially important regulatory step in MEKK3 signaling via dephosphorylation of Thr294, which reduces 14-3-3 binding correlating with MEKK3 pathway activation. The protein kinase MEKK3 is essential for tumor necrosis factor α (TNFα)- and lipopolysaccharide-induced activation of nuclear factor κB, although the mechanism by which TNF receptor 1 and Toll-like receptors regulate MEKK3 is largely unknown. In this study we have identified MEKK3 Thr294 as a novel site of phosphorylation that regulates MEKK3 binding with 14-3-3. Phosphorylation of MEKK3 at Thr294 was observed for both endogenous and ectopically expressed MEKK3. Mutation of Thr294 to alanine abolished 14-3-3-MEKK3 association and incubation with phosphorylated peptides mimicking Thr(P)294 competed for 14-3-3 binding. Mutation of Thr294 did not alter Ser526 phosphorylation within the activation loop. However, expression of T294A MEKK3 elevated TNFα-stimulated NF-κB transcriptional activity, suggesting that Thr294 phosphorylation and 14-3-3 binding negatively regulate MEKK3. Stimulation with TNFα or lipopolysaccharide caused a rapid decrease in Thr294 phosphorylation of endogenous MEKK3 and subsequent loss of 14-3-3 association. Thus, this study identifies a potentially important regulatory step in MEKK3 signaling via dephosphorylation of Thr294, which reduces 14-3-3 binding correlating with MEKK3 pathway activation. Protein kinases are key regulators of extracellular cues that illicit gene expression. One group of protein kinases involved in a wide array of cellular functions is the mitogen-activated protein kinase (MAPK) 2The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MAP(3)K, MAPK kinase kinase; MEKK, MAPK/ERK kinase kinase; TNF, tumor necrosis factor; LPS, lipopolysaccharide; IKK, IκB kinase; WT, wild type; CIP, calf intestinal phosphatase. family consisting of the well characterized extracellular regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (also called stress-activated protein kinase), p38 MAPK, and the Big MAPK/ERK5. These family members are all activated by linear phosphorylation cascades involving upstream protein kinases called MAPK kinase and MAP(3)K. Numerous MAP(3)K have been identified, including MEKK1, MEKK2, MEKK3, MEKK4, tumor progression locus 2, and transforming growth factor-B-activated kinase 1. MAP(3)Ks are emerging as important regulators of NF-κB. NF-κB belongs to a family of transcription factors that are essential mediators of pro-inflammatory signals involved in innate immunity. The mechanism of NF-κB regulation has been the subject of intense research for the past decade and is becoming better understood. NF-κB is sequestered to the cytoplasm by the inhibitor protein of κB(IκB). NF-κB can translocate to the nucleus following phosphorylation and degradation of IκBbyIκB kinase (IKK). Activation of IKK occurs rapidly following pro-inflammatory receptor activation, and both TNFα- and LPS-induced NF-κB activation is lost in mouse embryonic fibroblasts devoid of MEKK3 (1Yang J. Lin Y. Guo Z. Cheng J. Huang J. Deng L. Liao W. Chen Z. Liu Z.-G. Su B. Nat. Immunol. 2001; 2: 620-624Crossref PubMed Scopus (352) Google Scholar). IKK might be directly phosphorylated by MEKK3 (2Schmidt C. Peng B. Li Z. Sclabas G.M. Fujioka S. Niu J. Schmidt-Supprian M. Evans D.B. Abbruzzese J.L. Chiao P.J. Mol. Cell. 2003; 12: 1287-1300Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), but the mechanism of how TNF receptor 1 and TLR4 regulate MEKK3 phosphorylation of IKK in response to TNFα or LPS is not understood. A potential mechanism of MEKK3 regulation is through interaction with 14-3-3 molecules. 14-3-3 proteins are recognized as important cellular regulators by facilitating the interaction between phosphorylated proteins (3Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1195) Google Scholar, 4Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1353) Google Scholar, 5Tzivion G. Gupta V.S. Kaplun L. Balan V. Semin. Cancer Biol. 2006; 16: 203-213Crossref PubMed Scopus (194) Google Scholar) and are involved in regulating a diverse set of cellular proteins by altering activity, location, and stabilization (6Aitken A. Semin. Cancer Biol. 2006; 16: 162-172Crossref PubMed Scopus (646) Google Scholar, 7Yaffe M.B. FEBS Lett. 2002; 513: 53-57Crossref PubMed Scopus (557) Google Scholar). The interaction between another MAP(3)K, Raf-1, and 14-3-3 is an example of this complex regulation, with both activating and inactivating functions. For example, Raf-1 is inactivated by phosphorylation at Ser259, which promotes the association with 14-3-3 proteins and stabilizes Raf-1 in an inactive conformation that cannot be recruited and activated by Ras (8Roy S. McPherson R.A. Apolloni A. Yan J. Lane A. Clyde-Smith J. Hancock J.F. Mol. Cell Biol. 1998; 18: 3947-3955Crossref PubMed Scopus (116) Google Scholar, 9Light Y. Paterson H. Marais R. Mol. Cell Biol. 2002; 22: 4984-4996Crossref PubMed Scopus (102) Google Scholar, 10Clark G.J. Drugan J.K. Rossman K.L. Carpenter J.W. Rogers-Graham K. Fu H. Der C.J. Campbell S.L. J. Biol. Chem. 1997; 272: 20990-20993Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In contrast, Ser621 phosphorylation mediates distinct 14-3-3 binding, which stabilizes Raf-1 in an active conformation (11Tzivion G. Luo Z. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (390) Google Scholar, 12Thorson J.A. Yu L.W.K. Hsu A.L. Shih N.-Y. Graves P.R. Tanner J.W. Allen P.M. Piwnica-Worms H. Shaw A.S. Mol. Cell Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar, 13Ory S. Zhou M. Conrads T.P. Veenstra T.D. Morrison D.K. Curr. Biol. 2003; 13: 1356-1364Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). MEKK3 has been shown to associate with 14-3-3 (14Fanger G.R. Widmann C. Porter A.C. Sather S. Johnson G.L. Vaillancourt R.R. J. Biol. Chem. 1998; 273: 3476-3483Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 15Fritz A. Brayer K.J. McCormick N. Adams D.G. Wadzinski B.E. Vaillancourt R.R. J. Biol. Chem. 2006; 281: 6236-6245Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), although the site of interaction is not known, and therefore it is not clear what function 14-3-3 association might play. Here we show that 14-3-3 binding occurs at phosphorylated Thr294 of MEKK3. Agonists of the MEKK3 pathway, including TNFα and LPS, caused a reduction in Thr294 phosphorylation for endogenous MEKK3 leading to a loss of 14-3-3 binding. Furthermore, mutation of Thr294 to alanine led to elevated TNFα-stimulated NF-κB activation. Therefore, this study suggests that pro-inflammatory molecules such as TNFα and LPS contribute to MEKK3-dependent NF-κB activation by modulating Thr294 phosphorylation and 14-3-3 binding. Cell Culture—HEK 293, HeLa, and RAW 264.7 cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics at 37 °C, 5% CO2, and humidity. Plasmids and Mutagenesis—Human MEKK3 cDNA was cloned from IMAGE clone 7939519 by PCR and inserted into pCMV10-3xFLAG to introduce an N-terminal FLAG epitope. Mutagenesis of pCMV10-3xFLAG-MEKK3 was performed using QuikChange kit (Stratagene), and the mutations were sequence-verified. cDNA Transfection—HEK 293 or HeLa cells were plated onto 35-mm-diameter dishes at 80% confluency and transfected with 100-500 ng of cDNA using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. Transfection medium was removed and replaced with complete Dulbecco's modified Eagle's medium overnight. Cell Lysis, Immunoprecipitation, and Immunoblotting—The cells were lysed in 50 mm Tris-HCl, pH 7.4, 1% Triton X-100, 25 mm NaF, 25 mm β-glycerophosphate, 5 mm EDTA, 0.05% SDS, 100 nm okadaic acid and protease inhibitors. Anti-FLAG M2 antibody conjugated to agarose beads (Sigma) were added to lysates and incubated overnight at 4 °C. The beads were washed three times with lysis buffer, and proteins were eluted with 200 μl of lithium dodecyl sulfate sample buffer heated to 70 °C for 10 min. Portions of the lysates prior to immunoprecipitation were also boiled with lithium dodecyl sulfate-containing sample buffer. Lysates and immunoprecipitations were fractionated by SDS-PAGE. The proteins were transferred to a polyvinylidene difluoride membrane, blocked in 5% skim milk for 30 min, and probed with the appropriate antibody overnight at 4 °C. Secondary decoration with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies was performed at room temperature for 1.5 h. The proteins were visualized using ECL according to the manufacturers' protocol (Amersham Biosciences). For blotting using the Licor Odyssey infrared imager, the membranes were probed with IR700 anti-rabbit and IR800 anti-mouse secondary antibodies. FLAG was stained with anti-FLAG M2 mouse monoclonal (Sigma), and 14-3-3 isoforms were detected with anti-14-3-3β (H8; Santa Cruz Biotechnology). Metabolic Labeling—HEK 293 cells were plated onto 35-mm-diameter dishes at 80% confluency and transfected with 500 ng of pCMV10-FLAG-MEKK3 using Lipofectamine 2000 (Invitrogen) following the manufacturers' protocol. After 24 h, the cells were placed in phosphate-free Dulbecco's modified Eagle's medium with 1 mCi/ml 32P-labeled orthophosphate at 37 °C for 4 h. FLAG-MEKK3 was immunoprecipitated from detergent-solubilized lysates and fractionated on an 8% gel. 32P-Labeled MEKK3 was detected by autoradiography. Tryptic Digestion, Two-dimensional Phosphopeptide Mapping, and Phosphoamino Acid Analysis—Metabolically 32P-labeled FLAG-MEKK3 was isolated as described above, excised from the gel, and digested with 10 μg/ml tosylphenylalanyl chloromethyl ketone-treated trypsin (Promega) in 50 mm (NH4)HCO3, pH 7.8, overnight at 37 °C. The gel fragments were pelleted by centrifugation, and the remaining supernatant was transferred to clean tubes and dried under vacuum. Dried peptides were resuspended in 50 μl of performic acid, incubated on ice for 2 h, and dried under vacuum. The peptides were washed with diminishing volumes of water and resuspended in 5 μl of pH 1.9 electrophoresis buffer. Electrophoresis was performed on 200-μm microcrystalline cellulose plates (Kodak) at 1000 V, 7 °C for 30 min. The plates were chromatographed in the second dimension in chromatography buffer (n-butanol/pyridine/acetic acid/water, 32.5:25:5:20). The plates were dried, and phosphopeptides were visualized using an FX-Imager (Bio-Rad) or film autoradiography. If cold synthetic phosphopeptides were also run, were visualized with Phosphoamino acid was performed by 32P-labeled MEKK3 in 500 μl of heated to °C for min. The was removed under and the were washed with diminishing volumes of was performed on cellulose plates using buffer consisting of and 5% acid at at 7 °C for in and the was to were in a second dimension using pH buffer at at 7 °C for min. 32P-Labeled were detected by autoradiography. In of the 1 of a of and was also added that was visualized by Thr294 and to the Thr294 were by A was used as a Lysates FLAG-MEKK3 were incubated with 200 of or Anti-FLAG M2 agarose was and the were incubated 4 °C. The beads were washed three and proteins were eluted and by SDS-PAGE. 14-3-3 was detected using a anti-14-3-3β antibody In was immunoprecipitated from 293 cells and washed three times with buffer mm pH 7.4, mm β-glycerophosphate, mm NaF, 2 mm 100 nm okadaic acid, protease inhibitor and with kinase buffer mm pH 7.4, 25 mm 1 mm 2 mm 25 mm β-glycerophosphate, 1 μg/ml The were by and for at 30 °C. The were by an of sample buffer and The proteins were by transferred to polyvinylidene and with anti-FLAG M2 antibody or proteins were visualized by NF-κB cells were transfected with 500 ng of or with 500 ng of and 200 ng of After 24 h, the cells were with 10 ng TNFα for 4 h, and in the was of the were to MEKK3 for with better how MEKK3 is regulated by association with we the of 14-3-3 with MEKK3 with we that MEKK3 with or endogenous 14-3-3 isoforms detected the family of 14-3-3 proteins by with a 14-3-3 antibody a in all family interaction was for active MEKK3, the kinase did not 14-3-3 In within the of MEKK3 and for A. Brayer K.J. McCormick N. Adams D.G. Wadzinski B.E. Vaillancourt R.R. J. Biol. Chem. 2006; 281: 6236-6245Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, V. Huang J. Su B. J. 2006; PubMed Scopus Google Scholar), was also essential for 14-3-3 association mutation to alanine 14-3-3 that MEKK3 was for 14-3-3 as a of or of Thr294 as a of the site of 14-3-3 we by with and mutation of all to alanine did not MEKK3 The of 14-3-3 that MEKK3 be phosphorylated on that of we expressed MEKK3 and MEKK3 in cells with digested 32P-labeled MEKK3 with trypsin and the fragments by electrophoresis and cellulose chromatography A was in the MEKK3, suggesting that A was a that we performed acid on 32P-labeled MEKK3 a of and that MEKK3 was phosphorylated in cells on both did not phosphorylation was phosphorylation was and the MEKK3 to potential of The at within the MEKK3 of the MEKK3 was as a potential site of phosphorylation of the at the at and the at the phosphorylation for and also the 1 site for 14-3-3 protein binding. this site was phosphorylated in we in the the synthetic to the from The synthetic with the of this as The A were of the Thr294, between which in peptides of and with we that the from T294A MEKK3 that of were including A not of a Thr294 that Thr294 was a site of we a antibody to antibody from with the synthetic to we detected MEKK3 but not T294A MEKK3 In MEKK3 was with a reduction in phosphorylation at Thr294 observed by also MEKK3 phosphorylation at Thr294 by MEKK3 were detected with anti-FLAG phosphorylation of Thr294 was detected by MEKK3 stained for Thr(P)294 but were devoid in cells T294A MEKK3 that Thr294 was a site of phosphorylation of MEKK3. Thr294 14-3-3 that the loss of Thr294 phosphorylation of MEKK3 with the loss of 14-3-3 association. In we that the Thr294 within a potential 1 14-3-3 binding Thus, we the interaction of 14-3-3 with MEKK3 in which Thr294 was replaced with Mutation of Thr294 to alanine abolished 14-3-3 suggesting that this site an important in 14-3-3-MEKK3 that Thr294 was the site of 14-3-3 binding, we incubated MEKK3 prior to immunoprecipitation with phosphorylated or to the Thr294, to this with MEKK3 for 14-3-3 binding. The phosphorylated peptides abolished 14-3-3 the peptides on A phosphorylated as an and also did not 14-3-3-MEKK3 that Thr294 was a site of 14-3-3 protein of a Ser526 of Ser526 to alanine also abolished 14-3-3 Therefore, it that the of Thr294 phosphorylation on 14-3-3 association be a of Ser526 Ser526 we a antibody to this antibody from with the synthetic to detected MEKK3 but not MEKK3 Furthermore, MEKK3 was not detected by the with that phosphorylation of this site occurs by V. Huang J. Su B. J. 2006; PubMed Scopus Google Scholar). For MEKK3 the T294A Ser526 phosphorylated to a to 14-3-3 was abolished of Ser526 to a acid in a of Thr294 phosphorylation and 14-3-3 binding, with that kinase A. Brayer K.J. McCormick N. Adams D.G. Wadzinski B.E. Vaillancourt R.R. J. Biol. Chem. 2006; 281: 6236-6245Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, V. Huang J. Su B. J. 2006; PubMed Scopus Google Scholar). Therefore, we that Ser526 phosphorylation is and is for of Thr294 by MEKK3 but not directly in 14-3-3 association. by A. Brayer K.J. McCormick N. Adams D.G. Wadzinski B.E. Vaillancourt R.R. J. Biol. Chem. 2006; 281: 6236-6245Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) that Ser526 is to activity, and 14-3-3 binding to this site in from and that Ser526 of expressed MEKK3 was between and T294A MEKK3 cells were in the of mutation of Thr294 to alanine the Ser526 to activity, we lysed cells T294A MEKK3 and MEKK3 in the or of okadaic In the of okadaic acid, Ser526 phosphorylation of MEKK3 was by with MEKK3 lysed in the of okadaic acid, that Ser526 is to and family In to T294A MEKK3, we that Ser526 phosphorylation was MEKK3 was in the of okadaic acid MEKK3 at Thr294 and by TNFα and LPS have the phosphorylation of ectopically expressed MEKK3. to endogenous MEKK3 was phosphorylated at Thr294 and of MEKK3 including TNFα and LPS regulate the phosphorylation of this Thus, we immunoprecipitated MEKK3 from RAW 264.7 cells and probed with the The MEKK3 was with the Thr(P)294 suggesting that endogenous MEKK3 was phosphorylated at Thr294 blotting was performed using the Odyssey infrared and therefore we were to directly the of MEKK3 and Thr(P)294 on the with a for the of Thr294 phosphorylation of endogenous MEKK3. The of Thr(P)294 to MEKK3 with calf intestinal suggesting that endogenous MEKK3 is phosphorylated at The Thr(P)294 dephosphorylation by or a of antibody with MEKK3. we RAW 264.7 cells with which MEKK3 through activation TNF receptor 1. The cells were placed in medium for 4 and with infrared and of the Thr(P)294 to MEKK3 a decrease in with a decrease in Thr294 of following of A that the reduction in Thr294 phosphorylation was following TNFα and to by also the Toll-like receptor 4 LPS, which signaling including MEKK3 J. Lin Y. C. Cheng J. Liu Z.-G. Su B. Nat. Immunol. PubMed Scopus Google Scholar). LPS also caused a reduction of Thr294 phosphorylation by within 30 The decrease in phosphorylation of Thr294 with with and 2 phosphorylation 14-3-3-MEKK3 by we the dephosphorylation of Thr294 might the association of endogenous 14-3-3 with endogenous MEKK3. used LPS for LPS caused the decrease in Thr294 LPS of 14-3-3 was in with the decrease in Thr294 phosphorylation that the of Thr294 phosphorylation following LPS was to 14-3-3 binding. as we on the in the antibody be to with MEKK3. Thus, to that 14-3-3 was binding to endogenous MEKK3 at Thr294, we to with the to Thr294 the interaction of endogenous MEKK3 and endogenous but not a or a show that endogenous 14-3-3 with endogenous MEKK3 at and this is reduction of Thr294 In this set of we phosphorylation of Ser526 of endogenous MEKK3 was by LPS by V. Huang J. Su B. J. 2006; PubMed Scopus Google Scholar) that Ser526 phosphorylation LPS with antibody that Ser526 phosphorylation was elevated by following LPS the Odyssey infrared and the of to endogenous MEKK3 was by blotting on the MEKK3 with that the loss of 14-3-3 binding with the in Ser526 phosphorylation T294A MEKK3 NF-κB to with endogenous proteins that MEKK3 is phosphorylated at Thr294, and this phosphorylation in by TNFα or LPS The reduction in Thr294 phosphorylation the of endogenous 14-3-3 the loss of 14-3-3 binding with a in activation phosphorylation at Ser526 for endogenous MEKK3. therefore that the loss of 14-3-3 binding be involved in of MEKK3 MEKK3 was expressed in we that both Thr294 and Ser526 phosphorylation were phosphorylated to a and were not by TNFα or LPS is from endogenous MEKK3, the of MEKK3 and the of regulation are to how phosphorylation of Thr294 and binding to 14-3-3 might MEKK3 signaling in we performed in kinase MEKK3, T294A MEKK3, and MEKK3. the kinase MEKK3 did not the MEKK3 and T294A MEKK3 of that T294A MEKK3 not the of MEKK3. performed NF-κB to the activation of NF-κB in cells MEKK3 or T294A MEKK3 following to used the to that in pathway activation. was in cells MEKK3 or T294A MEKK3 but was by 4 of TNFα TNFα the of T294A NF-κB activation MEKK3 The were to an and the of expression of MEKK3 was between and T294A MEKK3 Thus, expression of the T294A MEKK3 elevated TNFα-stimulated NF-κB activation with MEKK3. The and MEKK3 protein kinases important in the activation of MAPK and NF-κB signaling following cellular and activation by pro-inflammatory For example, MEKK3 is essential for gene expression of TNF receptor and Toll-like receptor activation through the activation of c-Jun N-terminal p38 MAPK, and NF-κB (1Yang J. Lin Y. Guo Z. Cheng J. Huang J. Deng L. Liao W. Chen Z. Liu Z.-G. Su B. Nat. Immunol. 2001; 2: 620-624Crossref PubMed Scopus (352) Google Scholar, J. Lin Y. C. Cheng J. Liu Z.-G. Su B. Nat. Immunol. PubMed Scopus Google Scholar). The that and MEKK3 activation and are a subject of intense of signaling is through phosphorylation cascades that are regulated through association between signaling The well 14-3-3 in the and of J. C. S. L. A. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). 14-3-3 has been shown to with MEKK3, but the site of interaction has not been performed acid and with a antibody to Thr294 as a novel site of MEKK3 Mutation of this to alanine abolished 14-3-3 as did incubation of MEKK3 with a synthetic phosphorylated but not the Thr294 or a phosphorylated Phosphorylation of Thr294 was the of MEKK3, mutation of the at phosphorylation of Thr294 and the interaction between MEKK3 and 14-3-3. that phosphorylation of Thr294 by However, it is also that a kinase activated by MEKK3 phosphorylated For endogenous MEKK3, Thr294 to be phosphorylated and was with TNFα and The phosphorylation of endogenous MEKK3 detected by the Thr294 antibody be to MEKK3, which is by the shown in If this is the the decrease in Thr294 phosphorylation of endogenous MEKK3 be in with the that 14-3-3 binding is lost LPS Thr294 phosphorylation be to 14-3-3 from endogenous MEKK3. phosphopeptides mimicking Thr(P)294 competed with endogenous MEKK3 for endogenous 14-3-3 that in cells MEKK3, Ser526 phosphorylation was between MEKK3 and T294A MEKK3. However, we okadaic acid from the buffer, we that and T294A MEKK3 a reduction of Ser526 These are with the that Ser526 is to in cells A. Brayer K.J. McCormick N. Adams D.G. Wadzinski B.E. Vaillancourt R.R. J. Biol. Chem. 2006; 281: 6236-6245Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). However, in we not a between and T294A MEKK3 in the to dephosphorylation at we Ser526 phosphorylation of endogenous MEKK3, we a in Ser526 phosphorylation following with LPS, which was with the of V. Huang J. Su B. J. 2006; PubMed Scopus Google Scholar), described the of this Thus, is in with that Ser526 phosphorylation LPS and this the in Thr294 phosphorylation and the loss of 14-3-3 binding. suggests that dephosphorylation of MEKK3 at Thr294 and the subsequent loss of 14-3-3 binding might contribute to phosphorylation of Ser526 and be mechanism that might for that T294A MEKK3 NF-κB with MEKK3. The protein kinase to MEKK3 but might have distinct signaling functions and in the activation of NF-κB by regulating the activation of IKK and degradation of (2Schmidt C. Peng B. Li Z. Sclabas G.M. Fujioka S. Niu J. Schmidt-Supprian M. Evans D.B. Abbruzzese J.L. Chiao P.J. Mol. Cell. 2003; 12: 1287-1300Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). is to MEKK3 in acid the site of Thr294, suggesting that might associate with 14-3-3 at although this to be the is not to of with protein suggesting that the at is novel and to the The of this novel site and in MEKK3 an important in how the pro-inflammatory TNFα and the LPS regulate signals to gene expression. for

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