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...

Paeoniflorin (PF) is a monoterpene glycoside extracted from the root of Paeonia lactiflora Pall. Previous studies have demonstrated that PF inhibits the growth, invasion, and metastasis of tumors in vivo and in vitro. However, the effect of PF on hypoxia-induced epithelial–mesenchymal transition (EMT) in breast cancer cells remains unknown. Therefore, the objective of this study was to investigate the effect of PF on hypoxia-induced EMT in breast cancer cells, as well as characterize the underlying mechanism. The results presented in this study demonstrate that PF blocks the migration and invasion of breast cancer cells by repressing EMT under hypoxic conditions. PF also significantly attenuated the hypoxia-induced increase in HIF-1α level. Furthermore, PF prevented hypoxia-induced expression of phosphorylated PI3K and Akt in MDA-MB-231 cells. In conclusion, PF prevented hypoxia-induced EMT in breast cancer cells by inhibiting HIF-1α expression via modulation of PI3K/Akt signaling pathway. This finding provides evidence that PF can serve as a therapeutic agent for the treatment of breast cancer.

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