Tumor necrosis factor receptor-associated factor (TRAF) 4 is a new binding partner for the p70S6 serine/threonine kinase.
p70S6K is an intracellular serine/threonine kinase that mediates cell cycle progression and gene transcription. Immunofluorescent staining shows in factor-dependent hematopoietic M-07e cells that p70S6K is localized both in the cytosol and, after cytokine stimulation, also in the nucleus. We hypothesized that the p70S6K might interact with a transcription factor in the nucleus or with other proteins in the cytosol besides the S6 protein. By screening a yeast two-hybrid HeLa cDNA library with full-length p70S6K cDNA as bait, we identified tumor necrosis factor receptor-associated factor (TRAF) 4 as a new binding partner for this kinase. TRAF4 is a member of the TRAF family of putative signal-transducing proteins. Members of this family are capable of negatively regulating apoptotic pathways by inducing the expression of genes that promote cell survival. Immunoprecipitation experiments showed that stimulation of receptors of the tumor necrosis factor (TNF) family induced the formation of TRAF4/p70S6K complexes. Transfection studies showed that TRAF4 functions in p70S6K activation: TNF induced phosphorylation of S6, the main intracellular substrate of the kinase, in cells stably expressing TRAF4, but not in TRAF4-negative cells. In addition to its role in p70S6K activation, we postulate an anti-apoptotic role for TRAF4, because the agonistic anti-Fas antibody CH-11 induced apoptosis in untransfected HEK-293 cells, but not in TRAF4-expressing HEK-293 cells.


CONCLUSIONS
(i) TNF-receptor activation leads to activation of the p70S6K; (ii) TRAF4 is a mediator in this TNF-induced signaling pathway; and (iii) TRAF4 inhibits Fas-induced apoptosis.
Fleckenstein
Diana S
DS
Department of Human and Animal Cell Cultures, DSMZ-German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1B, 38124 Braunschweig, Germany.
Dirks
Wilhelm G
WG
Drexler
Hans G
HG
Quentmeier
Hilmar
H
D017209:Apoptosis; D002460:Cell Line; D002470:Cell Survival; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism* / Q000502:physiology; D020239:Receptor Cross-Talk; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism / Q000502:physiology; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism* / Q000502:physiology; D015398:Signal Transduction; D053449:TNF Receptor-Associated Factor 4; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D020798:Two-Hybrid System Techniques; D019014:fas Receptor / Q000502:physiology
D011506:Proteins; D018124:Receptors, Tumor Necrosis Factor; D053449:TNF Receptor-Associated Factor 4; C506367:TRAF4 protein, human; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D019014:fas Receptor; D038762:Ribosomal Protein S6 Kinases, 70-kDa