The forkhead-associated (FHA) domain is the only known phosphoprotein-binding domain that specifically recognizes phosphothreonine (pThr) residues, distinguishing them from phosphoserine (pSer) residues. In contrast to its very strict specificity toward pThr, the FHA domain recognizes very diverse patterns in the residues surrounding the pThr residue. For example, the FHA domain of Ki67, a protein associated with cellular proliferation, binds to an extended target surface involving residues remote from the pThr, whereas the FHA domain of Dun1, a DNA damage-response kinase, specifically recognizes a doubly phosphorylated Thr-Gln (TQ) cluster by virtue of its possessing two pThr-binding sites. The FHA domain exists in various proteins with diverse functions and is particularly prevalent among proteins involved in the DNA damage response. Despite a very short history, a number of unique structural and functional properties of the FHA domain have been uncovered. This review highlights the diversity of biological functions of the FHA domain-containing proteins and the structural bases for the novel binding specificities and multiple binding modes of FHA domains.

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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...

Cluster of differentiation (CD)69 is a leukocyte activation receptor involved in the maintenance of immune homeostasis and is positively selected in activated regulatory T (Treg) cells, implicating its role during Treg-cell differentiation. By RNA interference, we show that CD69 is not sufficient to support the conversion of CD4(+) naive T cells into Treg cells, whereas it does that of human peripheral blood mononuclear cells (hPBMCs) (P < 0.01), suggesting that a ligand-receptor interaction is required for CD69 function. Using immunoprecipitation and mass spectrometry, we identified the S100A8/S100A9 complex as the natural ligand of CD69 in hPBMCs. CD69 specifically associates with S100A8/S100A9 complex as confirmed by in vitro binding and competition assay, and the treatment of CD69 with peptide-N-glycosidase significantly abolishes such association. In agreement, the glycomics analysis determines the glycosylation site and the N-glycan composition of CD69, and terminal removal of sialic acid from that N-linked glycans reverses the generation of forkhead box P3-positive Treg cells (23.21%; P < 0.05). More specifically, we showed that CD69-S100A8/S100A9 association is required for the up-regulation of suppressor of cytokine signaling 3 resulting in inhibited signaling of signal transducer and activator of transcription 3 (36.54% increase upon CD69 silencing; P < 0.01). This might in turn support the secretion of key regulator TGF-β (∼ 3.28-fold decrease upon CD69 silencing; P < 0.05), leading to reduced production of IL-4 in hPBMCs. Our results demonstrate the functional and mechanistic interplays between CD69 and S100A8/S100A9 in supporting Treg-cell differentiation.