Homeostatic interactions between MEKK3 and TAK1 involved in NF-κB signaling

Several members of the mitogen-activated protein kinase kinase kinase (MAP3K) family including MEKK3 and TGFβ-activating kinase (TAK1) play nonredundant roles in activation of the NF-κB transcription factor. However, the mechanism by which MEKK3 mediates NF-κB signaling is not fully understood. In this report we investigate the association of murine MEKK3 with other proteins and their roles in NF-κB activation. Using tandem affinity purification TAK1 was identified as an endogenous protein that interacts with MEKK3. MEKK3–TAK1 interactions were confirmed by fluorescence resonance energy transfer and coimmunoprecipitation. MEKK3–TAK1 complexes contain non-phosphorylated forms of both molecules. Expression of non-phosphorylated TAK1 interferes with MEKK3 phosphorylation and NF-κB reporter activity induced by transient MEKK3 expression or TNFα stimulation. Addition of TAB1 facilitates TAK1 autophosphorylation and reverses the inhibitory effects of TAK1 on MEKK3 phosphorylation and NF-κB signal transduction in human 293 cells and TAK1 deficient mouse embryonic fibroblasts. The data provide insights into the homeostatic interactions that maintain basal NF-κB levels by holding the enzymes MEKK3 and TAK1 in their inactive state.