Involvement of TRAF4 in oxidative activation of c-Jun N-terminal kinase.
We previously found that the angiogenic factors TNFalpha and HIV-1 Tat activate an NAD(P)H oxidase in endothelial cells, which operates upstream of c-Jun N-terminal kinase (JNK), a MAPK involved in the determination of cell fate. To further understand oxidant-related signaling pathways, we screened lung and endothelial cell libraries for interaction partners of p47(phox) and recovered the orphan adapter TNF receptor-associated factor 4 (TRAF4). Domain analysis suggested a tail-to-tail interaction between the C terminus of p47(phox) and the conserved TRAF domain of TRAF4. In addition, TRAF4, like p47(phox), was recovered largely in the cytoskeleton/membrane fraction. Coexpression of p47(phox) and TRAF4 increased oxidant production and JNK activation, whereas each alone had minimal effect. In addition, a fusion between p47(phox) and the TRAF4 C terminus constitutively activated JNK, and this activation was decreased by the antioxidant N-acetyl cysteine. In contrast, overexpression of the p47(phox) binding domain of TRAF4 blocked endothelial cell JNK activation by TNFalpha and HIV-1 Tat, suggesting an uncoupling of p47(phox) from upstream signaling events. A secondary screen of endothelial cell proteins for TRAF4-interacting partners yielded a number of proteins known to control cell fate. We conclude that endothelial cell agonists such as TNFalpha and HIV-1 Tat initiate signals that enter basic signaling cassettes at the level of TRAF4 and an NAD(P)H oxidase. We speculate that endothelial cells may target endogenous oxidant production to specific sites critical to cytokine signaling as a mechanism for increasing signal specificity and decreasing toxicity of these reactive species.
Xu
You Cheng
YC
Department of Internal Medicine, University of Texas Southwestern and The Dallas Veterans Affairs Medical Center, Dallas, Texas 75216, USA.
Wu
Ru Feng
RF
Gu
Ying
Y
Yang
Yih-Sheng
YS
Yang
Meng-Chun
MC
Nwariaku
Fiemu E
FE
Terada
Lance S
LS
D000111:Acetylcysteine / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004730:Endothelium, Vascular / Q000502:physiology*; D004789:Enzyme Activation; D015723:Gene Library; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D008168:Lung; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism; D010084:Oxidation-Reduction; D010750:Phosphoproteins / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D053449:TNF Receptor-Associated Factor 4; D014162:Transfection; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D001616:beta-Galactosidase / Q000235:genetics
D000975:Antioxidants; D018076:DNA, Complementary; D010750:Phosphoproteins; D011506:Proteins; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D053449:TNF Receptor-Associated Factor 4; C506367:TRAF4 protein, human; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D001616:beta-Galactosidase; D000111:Acetylcysteine