Intermolecular Binding between TIFA-FHA and TIFA-pT Mediates Tumor Necrosis Factor Alpha Stimulation and NF-κB Activation

Abstract

T he forkhead-associated (FHA) domain, discovered in 1995 (10) and first suggested to bind phosphoproteins in 1998 (24), is known to specifically recognize phosphothreonine (pT) to exert its function (5,20). Although the sequence homology among different FHA-containing proteins is relatively low, the structural architecture of FHA domains is highly conserved. It contains a six-stranded ␤-sheet and another five-stranded ␤-sheet, forming a ␤-sandwich. The FHA-pT binding has been shown to regulate diverse biological functions, ranging from DNA damage repair to cell cycle checkpoints to signal transduction (18). Furthermore, the mechanism of FHA-phosphoprotein binding varies greatly among different FHA-containing proteins. The structure, specificity, mechanism, and biological functions of FHA domains have been summarized in recent reviews (16,18).TRAF-interacting protein with an FHA domain (TIFA) was first identified as a tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) binding protein. Consisting of 184 amino acids, TIFA is the smallest FHA domain-containing protein in humans (Fig. 1A). In the absence of TNF-␣ stimulation, TIFA overexpression in HEK 293T cells can activate NF-B and AP-1 (14), suggesting a direct involvement of TIFA in TNF-mediated immune responses. This involvement of TIFA was further attributed to the binding of TRAF2, which requires the TRAF domain of TRAF2 and almost the entire TIFA protein (residues 1 to 162) (14). TIFA was also reported to bind to TNF-associated factor 6 (TRAF6) (25). The consensus binding site of TIFA for TRAF6 was mapped to be glutamic acid 178 (E178) (11, 25), indicating different binding mechanisms in TIFA-TRAF2 and TIFA-TARF6 interactions. In addition, TIFA overexpression, even in the absence of interleukin-1 (IL-1), was shown to activate NF-B and c-Jun aminoterminal kinase (JNK), possibly through its enhancement of TRAF6 binding to IL-1 receptor-associated kinase 1 (IRAK-1). On the other hand, mutation of E178 abolished the binding of TIFA to TRAF6 and the ensuing activation of NF-B (25). In a follow-up report, TIFA was shown to promote oligomerization and ubiquitination of TRAF6, leading to activation of IB kinase (IKK), based on in vitro studies (6).Although the studies of Takatsuna et al. (25) and Ea et al. (6) have previously established the key function of TIFA in its interaction with TRAF6, several issues still remain inconclusive. For example, TIFA has been suggested to be phosphorylated, and the integrity of the FHA domain of TIFA is essential for its function (6, 25), but little information has been unveiled about the molecular basis of TIFA phosphorylation and its functional consequences.In this work, we report that threonine 9 (T9) is a newly identified phosphorylation site of TIFA and that the phosphorylation level of T9 increases upon TNF-␣ treatment. Based on data collected here, we concluded that TIFA-FHA binds to this pT9 site. Such a TIFA-FHA/pT9 binding directs TIFA self-association and promotes NF-B activation through the oligomerizatio...

Aurora A and NF-κB Survival Pathway Drive Chemoresistance in Acute Myeloid Leukemia via the TRAF-Interacting Protein TIFA

“…Our previous study uncovered that this functional event requires the phospho-threonine 9 (pThr9)-directed oligomerization of TIFA dimer upon TNFa stimulation (21). In the current study, we further identified Aurora A as an essential kinase for TIFA phosphorylation at Thr9 and showed that TIFA mediates the Aurora A-dependent NF-kB signaling pathway.…”

“…All resulting STR profiles were matched with known ATCC fingerprints (www.ATCC.org). Maintenance of respective cell lines followed our previous protocols (21)(22)(23). 293T and HeLa cells were maintained in DMEM(Gibco).…”

“…Experiments followed protocols we previously established (21). 293T cells under indicated treatment were lysed with CHAPS lysis buffer (20 mmol/L PIPES, 1 mmol/L Na 3 VO 4 , 1 mmol/L EGTA, 50 mmol/L Tris-HCl, 150 mmol/L NaCl, 50 mmol/L NaF, 1% CHAPS, and 10% glycerol) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitor (Sigma).…”

“…TIFA-specific mAb was raised as described previously (21). Anti-Aurora A, antiphospho-Aurora A (Thr288), anti-Bcl-2, and anti-Bcl-X L were from Cell Signaling Technology.…”

“…Immunofluorescence staining experiment was performed as described previously with minor modifications (21). Subconfluent HeLa cells seeded on coverslips were treated with 100 ng/mL nocodazole (Sigma) or DMSO for 16 hours to induce G 2 -M arrest, followed by treatment with PBS or 10 ng/mL TNFa (R&D Systems) for 30 minutes.…”