In addition to inflammatory responses, TRAF proteins regulate cell proliferation and survival. TRAF family members share a stretch of conserved sequence at their C-terminal domains known as the TRAF domain (Rothe et al. 1994). The TRAF domain is divided into a highly conserved carboxyl terminal region (C-domain), the TRAF-C sub-domain, and the coiled-coil amino terminal region and the TRAF-N sub-domain (Inoue et al. 2000). The TRAF domain mediates homo- and hetero-dimerization of TRAF family members, although TRAF4 shows a poor ability to associate with the other family members. The TRAF domain confers also direct or indirect interaction with the intracellular domain of cell surface receptors and signal transducers (Kaufman and Choi 1999; Cheng et al. 1995).
All TRAFs, except TRAF1, contain an amino terminal RING finger motif, in addition to the TRAF domain (Hsu et al. 1996). TRAF4 contains seven zinc finger domains, while TRAF2, TRAF3, TRAF5, and TRAF6 have fewer zinc finger domains.
TRAF6 (gene accession number of TRAF6 NM_004620, http://www.ncbi.nlm.nih.gov/gene/7189) was first identified independently as an adaptor protein, important for NF-κB activation, initiated by IL-1 and CD40. It was initially isolated using yeast two hybrid systems using an EST expression library (Cao et al. 1996; Ishida et al. 1996). TRAF6 is a cytosolic protein although it is predominantly present in membrane-bound cellular compartments (Dadgostar and Cheng 2000). TRAF6 contains a C3HC3D-type Really Interesting New Gene (RING) finger followed by five zinc finger regions in its N-terminus (Inoue et al. 2000). TRAF6 plays a specific role in innate and adaptive immune response apart from another diverse range of physiological processes (Naito et al. 1999; Lomaga et al. 1999). TRAF6 exists in a trimeric form at very high concentrations. TRAF6 null mice have an abnormal phenotype of defective bone formation and die at early age (Naito et al. 1999; Lomaga et al. 1999).
TRAF6 functions as an E3 ubiquitin ligase that interacts with the E2-conjugating enzyme Msm2, which consists of Ubc13 and Uev1A to synthesize lysine-63-linked polyubiquitin (Deng et al. 2000; Wang et al. 2001). The enzymatic activity of TRAF6 is promoted by its oligomerization and autoubiquitination on Lys124, which has been reported to be the key ubiquitin lysine acceptor site on TRAF6 (Lamothe et al. 2007; Bhoj and Chen 2009). Previously it has been reported that TRAF6 interacts with tumor members of the TNFR family such as CD40 and RANK, IL-1R/Toll-like receptor (TLR) family members (Muzio et al. 1997; Wesche et al. 1999; Suzuki et al. 2002). Recent studies have demonstrated that TRAF6 also binds to the type I transforming growth factor β (TGFβ) receptor (TβRI) (Sorrentino et al. 2008).
Role of TRAF6 for Smad-Independent TGFβ Signaling
TGFβ is known to transduce signals through Smad proteins as well as non-Smad mediators, like extracellular signal-regulated kinases (ERKs), the small GTPases Rho, Rac, Cdc42, c-Jun N-terminal kinases (JNKs), and mitogen-activated protein kinase (MAPK) (Derynck and Zhang 2003; Landstrom 2010; Mu et al. 2011a). Yamaguchi et al. (1995) originally showed that transforming growth factor-β-activated kinase-1 (TAK1), a member of mitogen-activated protein kinase kinase kinase (MAPKKK) family, functions as a mediator in a signaling pathway of TGFβ superfamily members. TAK1 regulates various cellular responses like cell survival through activation of JNK, p38 MAPK, and inflammatory responses via IκB kinase (IKK) and NfKappaB pathways (Adhikari et al. 2007; Thakur et al. 2009; Landstrom 2010 plus Hamidi et al. 2012). TAK1 is also crucial for the activation of LKB1 serine/threonine kinase which in turn controls cell metabolism, growth, and polarity (Adhikari et al. 2007; Shaw 2009; Thakur et al. 2009; Landstrom 2010; Mu et al. 2011a).
Later, Sorrentino et al. (2008) and Yamashita et al. (2008) reported that TRAF6 is crucial for the activation of downstream targets p38 MAPK pathway in TGFβ signaling cascade, through its enzymatic activity as an E3 ligase. Sorrentino et al. showed that TRAF6 constitutively binds to TGFbeta type I receptor conserved consensus motif (basic residue-X-P-X-E-X-X-aromatic/acidic acid) TGF-β type 1 receptor (TβR1), and this interaction leads to the autoubiquitination of TRAF6, in response to TGFβ stimulation of cells which in turn causes Lys63-linked polyubiquitination of TAK1 at Lys34 (Sorrentino et al. 2008). The kinase activity of TAK1 might be inhibited by N-terminal part of TAK1 itself (Yamaguchi et al. 1995); therefore, it is possible that this cascade of events then leads to the activation of TAK1 either due to a conformational change to open up its structure or recruitment of the TAK1-binding proteins 2 and 3 (TAB2 and TAB3) (Sorrentino et al. 2008; Landstrom 2010). Kim et al. (2009) reported that TGFβ-induced autoubiquitination of TAK1 recruits the adaptor molecule TAB1.
Recently, Mu et al. (2011b) showed a novel TRAF6-dependent pathway by which TGFβ mediates its oncogenic effects. TRAF6 binds to TβRI via conserved consensus site and upon TGFβ-induced autoubiquitination and activation causes Lys63-linked polyubiquitination of TβRI. In cancer cells, TβRI undergoes cleavage by TNF-alpha converting enzyme (TACE) in a PKCζ-dependent manner, and the intracellular domain (ICD) of cleaved TβRI translocates to the nucleus where it associates with the transcriptional regulator p300 and the snail promoter (Fig. 2). The association of TβRI-ICD to p300 and the snail promoter enhances tumor invasion by induction of pro-invasive genes such as snail and MMP2. The negative regulation of TGFβ signaling due to the ectodomain shedding of TβRI by TACE was previously reported (Liu et al. 2009). Their report suggests that the TACE-mediated ectodomain shedding of TβRI also results in reduced TGFβ-mediated growth inhibition and EMT.
Our group have also found that TβRI undergoes regulated intramembrane proteolysis in a manner dependent on TRAF6, resulting in the liberation of the intracellular domain (ICD) of TβRI in cancer cells (Gudey et al. 2014a). TRAF6 was found to recruit Presenilin1 (PS1), a key regulator protein in the γ-secretase complex to TβRI. TRAF6 activates PS1 by promoting Lys63-linked polyubiquitination of PS1. Activated PS1 cleaved TβRI in the transmembrane domain, and the generated TβRI-ICD enters the nucleus where it was found to bind to the promoter of TβRI encoding gene. The TRAF6- and PS1-induced cleavage and nuclear localization of TβRI promoted TGFβ-induced invasion in cancer cells (Gudey et al. 2014a, b). In the report from Sundar et al., we provided evidence for that TRAF6-promoted Lys63-linked polyubiquitination of Lys178 in TβRI (Sundar et al. 2015). TRAF6-mediated Lys63-linked polyubiquitination of TβRI at Lys178 was shown to promote regulation of genes controlling the cell cycle (CCND1), differentiation, and invasiveness of prostate cancer cells (Sundar et al. 2015).
Recently, Song et al. reported that the endocytic adaptor proteins APPL1 and APPL2 are required for TRAF6-mediated TGFβ-induced nuclear translocation of TβRI-ICD in human prostate and breast cancer cell lines. TRAF6 promoted TβRI-APPL1 complex formation and Lys63-linked polyubiquitination of APPL1 (Song et al. 2016). Moreover, APPL1-TβRI-ICD complexes were found at high levels in aggressive human prostate cancer tissue. Our group also reported that the adaptor protein CIN85 promoted recycling of TβRI to the cell surface by interaction of TβRI with the SH3 domain of CIN85 in response to TGFβ in a TRAF6-dependent manner (Yakymovych et al. 2015). Sitaram et al. reported that the generation of ALK5-ICD is positively associated with canonical TGFβ signaling and has a key role in promoting aggressiveness and invasion of clear cell renal cell carcinoma (ccRCC) (Sitaram et al. 2016).
RanBPM, a scaffold protein, was identified as a novel binding partner of TRAF6 and TBRI. Overexpression of RanBPM blocked TGFb-induced nuclear accumulation of TBRI-ICD (Zhang et al. 2014). Smad6 controls activation of the TβRI-TRAF6 pathway. Smad6 negatively regulates TGFβ-induced non-canonical TGFβ-TRAF6-TAK1-p38 MAPK pathway. Smad6 recruits deubiquitinating enzyme, A20, resulting in deubiquitination of TRAF6 in a TGFβ-dependent manner (Jung et al. 2013).
The role of TRAF6 in other signaling cascades besides TGFβ has recently been further explored. TRAF6 has been shown to promote Lys63-linked polyubiquitination of APPL1, in response to insulin hepatocytes, by causing membrane translocation of Akt (Cheng et al. 2013). TRAF6 is also reported to act as an essential molecular switch for the development of cardiac hypertrophy (Ji et al. 2016). This is dependent on TRAF6 autoubiquitination and its binding to TAK1, which in turn cause ubiquitination of TAK1 by TRAF6. High expression levels of TRAF6 were found in esophageal cancer tissues, and patients with high TRAF6 expression have significantly shorter survival time (Liu et al. 2016). Another study shows that miR-146b-5p inhibited the proliferation of glioma cells and promoted apoptosis by directly targeting TRAF6 (Liu et al. 2015). They found that TRAF6 overexpression was associated with miR-146b-5p downregulation and poor prognosis in human gliomas.
TRAF6 is an important enzyme causing Lys63-linked polyubiquitination of its substrates to promote pro-inflammatory responses downstream of members of the TNF-R, IL-1R, TLR, and TGFβ receptors. Recently, TRAF6 was also identified to be a crucial regulatory enzyme for Lys63-linked activations of TAK1 and for the proteolytic cleavage of TβRI by TACE and PS1. The TβRI-ICD enters the nucleus via its association with the endosomal adaptor protein APPL1. The nuclear TβRI-ICD promotes invasion of cancer cells. The identification of TRAF6 as a key regulator of TGFβ-induced tumor invasion will hopefully help to design novel inhibitors to prevent tumorigenic effects.
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