T-cell regulator RNF125/TRAC-1 belongs to a novel family of ubiquitin ligases with zinc fingers and a ubiquitin-binding domain

Department of Immunobiology, King's College London, 2nd Floor New Guy's House, Guy's Hospital, St Thomas Street, London SE1 9RT, UK.
Biochemical Journal (Impact Factor: 4.4). 03/2008; 410(1):101-11. DOI: 10.1042/BJ20070995
Source: PubMed

ABSTRACT The recently identified RNF125 [RING (really interesting new gene) finger protein 125], or TRAC-1 (T-cell RING protein in activation 1), is unique among ubiquitin ligases in being a positive regulator of T-cell activation. In addition, TRAC-1 has been shown to down-modulate HIV replication and to inhibit pathogen-induced cytokine production. However, apart from the presence of an N-terminal C3HC4 (Cys(3)-His-Cys(4)) RING domain, the TRAC-1 protein remains uncharacterized. In the present paper, we report novel interactions and modifications for TRAC-1, and elucidate its domain organization. Specifically, we determine that TRAC-1 associates with membranes and is excluded from the nucleus through myristoylation. Our data are further consistent with a crucial role for the C-terminus in TRAC-1 function. In this region, novel domains were recognized through the identification of three closely related proteins: RNF114, RNF138 and RNF166. TRAC-1 and its relatives were found to contain, apart from the RING domain, a C2HC (Cys(2)-His-Cys)- and two C2H2 (Cys(2)-His(2))-type zinc fingers, as well as a UIM (ubiquitin-interacting motif). The UIM of TRAC-1 binds Lys(48)-linked polyubiquitin chains and is, together with the RING domain, required for auto-ubiquitination. As a consequence of auto-ubiquitination, the half-life of TRAC-1 is shorter than 30 min. The identification of these novel modifications, interactions, domains and relatives significantly widens the contexts for investigating TRAC-1 activity and regulation.

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Available from: Ana Giannini, Nov 14, 2014
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    • "Among those, we found UPF1, PABPC1, and PABPC4 to be interacting with MOV10 in an RNA-independent manner. In addition, we identified RNF166 belonging to a subfamily of RING ubiquitin ligases (Giannini et al., 2008) and the DEAD box RNA helicase eIF4A3, and the inhibitor of Moloney murine and HIV-1 viruses ZC3HAV1/ZAP (Zhu et al., 2011) as RNA-independent interactors. To confirm the interaction between MOV10 and UPF1, we performed reverse IP experiments using FLAG/HA-tagged UPF1 as bait and identified MOV10 as enriched in both crossover label-swap experiments (Figure 4C and Table S5). "
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    ABSTRACT: RNA helicases are important regulators of gene expression that act by remodeling RNA secondary structures and RNA-protein interactions. Here, we demonstrate that MOV10 has an ATP-dependent 5' to 3' in vitro RNA unwinding activity and determine the RNA-binding sites of MOV10 and its helicase mutants using PAR-CLIP. We find that MOV10 predominantly binds to 3' UTRs upstream of regions predicted to form local secondary structures and provide evidence that MOV10 helicase mutants are impaired in their ability to translocate 5' to 3' on their mRNA targets. MOV10 interacts with UPF1, the key component of the nonsense-mediated mRNA decay pathway. PAR-CLIP of UPF1 reveals that MOV10 and UPF1 bind to RNA in close proximity. Knockdown of MOV10 resulted in increased mRNA half-lives of MOV10-bound as well as UPF1-regulated transcripts, suggesting that MOV10 functions in UPF1-mediated mRNA degradation as an RNA clearance factor to resolve structures and displace proteins from 3' UTRs.
    Molecular cell 04/2014; 54(4). DOI:10.1016/j.molcel.2014.03.017 · 14.02 Impact Factor
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    • "However, the precise mechanisms underlying this positional effect remain yet to be elucidated. Also, as the function of RNF166 in vivo is poorly understood, we can only speculate that the protein could constitute a link between TH and the regulation of ubiquitin ligation [35]. Nevertheless, the example of RNF166 demonstrates that mice expressing a tagged nuclear receptor isoform can be used for identifying novel DNA-binding sites in vivo, while circumventing the need for specific antibodies or artificial expression systems. "
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    ABSTRACT: Thyroid hormone receptor alpha 1 (TRα1) is well recognized for its importance in brain development. However, due to the difficulties in predicting thyroid hormone response elements (TREs) in silico and the lack of suitable antibodies against TRα1 for chromatin immunoprecipitation, only a few direct TRα1 target genes have been identified in the brain. Here we demonstrate that mice expressing a TRα1-GFP fusion protein from the endogenous TRα locus provide a valuable animal model to identify TRα1 target genes. To this end, we analyzed DNA-TRα1 interactions in vivo using chromatin immunoprecipitation with an anti-GFP antibody. We validated our system using established TREs from neurogranin and hairless, and by verifying additional TREs from known TRα1 target genes in brain and heart. Moreover, our model system enabled the identification of novel TRα1 target genes such as RNF166. Our results demonstrate that transgenic mice expressing a tagged nuclear receptor constitute a feasible approach to study receptor-DNA interactions in vivo, circumventing the need for specific antibodies. Models like the TRα1-GFP mice may thus pave the way for genome-wide mapping of nuclear receptor binding sites, and advance the identification of novel target genes in vivo.
    Bioscience Reports 02/2013; 33(2). DOI:10.1042/BSR20120124 · 2.64 Impact Factor
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    • "Deletion of the UIM domain also does not affect MEKK1-dependent JNK activation, apoptosis or modification in our B cell reconstitution system. UIM regions contained in other proteins have been shown to directly interact with ubiquitin and can enhance the ubiquitination of these UIM-containing proteins [16], [25]. For example, auto-ubiquitination of TRAC-1 requires both its RING and UIM domains, as point mutations in either domain reduce the amount of ubiquitinated TRAC-1 [25]. "
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    ABSTRACT: MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the MAPK JNK and is required for microtubule inhibitor-induced apoptosis in B cells. Here, we find that apoptosis induced by actin disruption via cytochalasin D and by the protein phosphatase 1/2A inhibitor okadaic acid also requires MEKK1 activation. To elucidate the functional requirements for activation of the MEKK1-dependent apoptotic pathway, we created mutations within MEKK1. MEKK1-deficient cells were complemented with MEKK1 containing mutations in either the ubiquitin interacting motif (UIM), plant homeodomain (PHD), caspase cleavage site or the kinase domain at near endogenous levels of expression and tested for their sensitivity to each drug. We found that both the kinase activity and the PHD domain of MEKK1 are required for JNK activation and efficient induction of apoptosis by drugs causing cytoskeletal disruption. Furthermore, we discovered that modification of MEKK1 and its localization depends on the integrity of the PHD.
    PLoS ONE 02/2011; 6(2):e17310. DOI:10.1371/journal.pone.0017310 · 3.23 Impact Factor
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