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

J Proteome Res. 2010 Jul 2;9(7):3465-78. doi: 10.1021/pr9011377.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • 14-3-3 Proteins / genetics
  • 14-3-3 Proteins / metabolism*
  • ATPases Associated with Diverse Cellular Activities
  • Carrier Proteins / metabolism
  • Cell Line
  • Computational Biology / methods*
  • DNA Helicases / metabolism
  • Humans
  • Immunoblotting
  • MAP Kinase Kinase Kinases / metabolism
  • MAP Kinase Signaling System / physiology*
  • Microscopy, Confocal
  • NF-kappa B / metabolism*
  • Oligopeptides
  • Peptides / genetics
  • Peptides / metabolism
  • Phosphoprotein Phosphatases / metabolism
  • Protein Interaction Mapping / methods*
  • Protein Phosphatase 2C
  • Reproducibility of Results
  • Tumor Necrosis Factor-alpha / metabolism*

Substances

  • 14-3-3 Proteins
  • Carrier Proteins
  • NF-kappa B
  • Oligopeptides
  • Peptides
  • Tumor Necrosis Factor-alpha
  • YWHAE protein, human
  • FLAG peptide
  • MAP Kinase Kinase Kinases
  • MAP kinase kinase kinase 7
  • PPM1A protein, human
  • PPM1B protein, human
  • PPM1G protein, human
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 2C
  • ATPases Associated with Diverse Cellular Activities
  • DNA Helicases
  • RUVBL2 protein, human