Intracellular localization and transcriptional regulation of tumor necrosis factor (TNF) receptor‐associated factor 4 (TRAF4)

To gain insight in the subcellular localization of tumor necrosis factor receptor-associated factor (TRAF4) we analyzed GFP chimeras of full-length TRAF4 and various deletion mutants derived thereof. While TRAF4–GFP (T4–GFP) was clearly localized in the cytoplasm, the N-terminal deletion mutant, T4(259–470), comprising the TRAF domain of the molecule, and a C-terminal deletion mutant consisting mainly of the RING and zinc finger domains of TRAF4 were both localized predominantly to the nucleus. Passive nuclear localization of T4(259–470) can be ruled out as the TRAF domain of TRAF4 was sufficient to form high molecular weight complexes. T4(259–470) recruited full-length TRAF4 into the nucleus whereas TRAF4 was unable to change the nuclear localization of T4(259–470). Thus, it seems that individual T4(259–470) mutant molecules are sufficient to direct the respective TRAF4–T4(259–470) heteromeric complexes into the nucleus. In cells forming cell–cell contacts, TRAF4 was recruited to the sites of contact via its C-TRAF domain. The expression of some TRAF proteins is regulated by the NF-κB pathway. Thus, we investigated whether this pathway is also involved in the regulation of the TRAF4 gene. Indeed, in primary T-cells and Jurkat cells stimulated with the NF-κB inducers TNF or phorbol 12-myristate 13-acetate (PMA), TRAF4-mRNA was rapidly up-regulated. In Jurkat T-cells deficient for I-κB kinase γ (IKKγ, also known as NEMO), an essential component of the NF-κB-inducing–IKK complex, induction of TRAF4 was completely inhibited. In cells deficient for RIP (receptor interactive protein), an essential signaling intermediate of TNF-dependent NF-κB activation, TNF-, but not PMA-induced up-regulation of TRAF4 was blocked. These data suggest that activation of the NF-κB pathway is involved in up-regulation of TRAF4 in T-cells.