Phosphorylation of MEKK3 at threonine 294 promotes 14-3-3 association to inhibit nuclear factor kappaB activation.
The protein kinase MEKK3 is essential for tumor necrosis factor alpha (TNFalpha)- and lipopolysaccharide-induced activation of nuclear factor kappaB, although the mechanism by which TNF receptor 1 and Toll-like receptors regulate MEKK3 is largely unknown. In this study we have identified MEKK3 Thr(294) as a novel site of phosphorylation that regulates MEKK3 binding with 14-3-3. Phosphorylation of MEKK3 at Thr(294) was observed for both endogenous and ectopically expressed MEKK3. Mutation of Thr(294) 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 Thr(294) did not alter Ser(526) phosphorylation within the activation loop. However, expression of T294A MEKK3 elevated TNFalpha-stimulated NF-kappaB transcriptional activity, suggesting that Thr(294) phosphorylation and 14-3-3 binding negatively regulate MEKK3. Stimulation with TNFalpha or lipopolysaccharide caused a rapid decrease in Thr(294) 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 Thr(294), which reduces 14-3-3 binding correlating with MEKK3 pathway activation.
Matitau
Adi E
AE
Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada.
Scheid
Michael P
MP
D048948:14-3-3 Proteins / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002384:Catalysis; D002460:Cell Line; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D048748:MAP Kinase Kinase Kinase 3 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D048948:14-3-3 Proteins; D000906:Antibodies; D008070:Lipopolysaccharides; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D013912:Threonine; D048748:MAP Kinase Kinase Kinase 3