14-3-3 Epsilon Dynamically Interacts with Key Components of Mitogen-activated Protein Kinase Signal Module for Selective Modulation of the TNF-alpha-induced Time Course-dependent NF-kappaB Activity

Inflammation is tightly regulated by nuclear factor-kappa B (NF-kappaB), and if left unchecked excessive NF-kappaB activation for cytokine overproduction can lead to various pathogenic consequences including carcinogenesis. A proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), can be used to explore possible mechanisms whereby unknown functional pathways modulate the NF-kappaB activity for regulating TNF-alpha-induced inflammation. Given the multifunctional nature of 14-3-3 family proteins and the recent finding of their presence in the TNF-alpha/NF-kappaB pathway network, we used a dual-tagging quantitative proteomic method to first profile the TNF-alpha-inducible interacting partners of 14-3-3 epsilon, the least characterized 14-3-3 isomer in the family. For the first time, we found that TNF-alpha stimulation enhances the interactions between 14-3-3 epsilon and some key components in the mitogen-activated protein kinase (MAPK) signal module which is located at the immediate upstream of NF-kappaB, including transforming growth factor-beta activated kinase-1 (TAK1) and its interacting protein, protein phosphatase 2C beta (PPM1B). By using confocal laser scanning, we observed the TNF-alpha-induced colocalizations among 14-3-3 epsilon, TAK1, and protein phosphatase 2C beta (PPM1B), and these interactions were also TNF-alpha-inducible in different cell types. Further, we found that during the full course of the cellular response to TNF-alpha, the interactions between 14-3-3 epsilon and these two proteins were dynamic and were closely correlated with the time course-dependent changes in NF-kappaB activity, suggesting that these 14-3-3 epsilon interactions are the critical points of convergence for TNF-alpha signaling for modulating NF-kappaB activity. We then postulated a mechanistic view describing how 14-3-3 epsilon coordinates its dynamic interactions with TAK1 and PPM1B for differentially modulating TNF-alpha-induced changes in NF-kappaB activity. By using bioinformatics tools, we constructed the network involving most of the 14-3-3 epsilon interacting proteins identified in our proteomic study. We revealed that 14-3-3 epsilon coordinates the cross talks between the MAPK signal module and other molecular pathways/biological processes primarily including protein metabolism and synthesis, DNA repair, and cell cycle regulation where pharmacological targets for therapeutic intervention could be systematically located.