Phosphorylation of MEKK3 at threonine 294 promotes 14-3-3 association to inhibit nuclear factor κB activation

Abstract

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 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 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. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.