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  • The murine subfamily members including and have been identif

    2019-08-13

    The murine subfamily members including , , , and have been identified and their encoded proteins showed deubiquitinating enzymatic activity , , , . It has been demonstrated that is an immediate-early gene induced by IL-3 in B-lymphocytes and is an IL-2 specific immediate-early gene in T-lymphocytes . Recently, we identified a novel gene that is expressed not only in T-lymphocytes, but also in embryonic stem cells and myeloid cells . This suggests that at least several deubiquitinating enzymes may exist in mice. These Dubs (Dub-1, Dub-1A, Dub-2, and Dub-2A) are classified as a new Dub subfamily of deubiquitinating enzymes , , , . They are very similar throughout their primary amino tcy sequences (), but their molecular weights are different (Dub-1, Dub-1A, Dub-2, and Dub-2A are 59, 51, 62, and 64kDa, respectively) , . While the site-directed mutagenesis experiments for the conserved amino acids of Dub-1, Dub-1A, Dub-2, and Dub-2A showed that they are required for deubiquitinating enzyme activity, the deletion analysis of C-terminal region for them revealed no effect for the deubiquitinating enzyme activity , . Therefore, we suggest that the C-terminal region of these enzymes may be involved in substrate specificity and/or localization. The murine gene contains an IL-3-inducible enhancer element that is activated in a Jak2-dependent, Stat5-independent manner . This enhancer element includes two AP-1 sites, two GATA sites, an Ets site, and a CBF site. Interestingly, transcriptional reporter assays have previously shown that two AP-1 sites, an Ets site, and a GATA site at the N-terminus, but not a GATA site at the C-terminus and a CBF site, are required for cytokine inducibility . In addition, gel shift assays revealed that IL-3 activates the association of an AP-1 complex containing JunD with the AP-1 sites and the association of another protein complex with the Ets motif . It was striking to find that there is considerable base pair identity in enhancer regions of , , and , suggesting the conservation of enhancer function , , . The enhancer region contains an Ets site, two AP-1 sites, and a GATA site and we demonstrated that the enhancer activity of is comparable to the activity of enhancer . Therefore, it is possible for the subfamily members of deubiquitinating enzyme gene to contain conserved motifs in the enhancer domain to be expressed in lymphocytes. In this study, we investigated cytokine inducibility for binding motifs in the enhancer domain of by transcriptional reporter assays whether binding motifs showed similar cytokine inducibility as shown in those of and . We also analyzed conserved amino acids of Dub-2A for deubiquitinating enzyme activity by site-directed mutagenesis followed by in vivo and in vitro deubiquitinating enzyme assays to compare with other UBPs including Dub-1, Dub-1A, and Dub-2. Materials and methods
    Results
    Discussion Several deubiquitinating enzymes have been recently shown to regulate cell growth. This includes the mammalian tre-2 oncoprotein that shows transforming activity in NIH3T3 fibroblasts, the murine UNP gene that is tumorigenic in nude mice, and the Drosophila FAF gene that determines cell growth and differentiation during Drosophila eye development [27], [28]. Since it has been known that both DUB-1 and DUB-2 are immediate-early genes and Dub-1 enzyme regulates cell growth, we expect that they get expressed very transiently and function as deubiquitinating enzymes in response to cytokine stimuli. Thus, their expression may be correlated to their function during cell cycle progression, antigen presentation, signal transduction, transcriptional activation, and DNA repair. Therefore, finding the specific substrates for each Dub enzyme will help to understand their physiological roles in response to cytokine stimuli in vivo. It is interesting that deubiquitinating enzymes have dual functions; rescue protein targets from degradation by cleaving ubiquitin before proteasome entrance, and accelerate the degradation by cleaving ubiquitin from peptide remnants after proteasome passage [13], [27]. However, relatively little information is available on deubiquitinating enzymes compared with ubiquitinating enzymes. There are five classes of deubiquitinating enzymes, UCHs, UBPs, JAMMs, ULPs, and OUT-domain Ub-aldehyde-binding protein (OTUs) [4], [11], [12]. It has been demonstrated that these enzymes contain a putative catalytic triad which consisted of cysteine, aspartic acid, and histidine residues [13], [15]. In this study, we found that the conserved Asp at amino acid 323 was not required for the catalytic activity of Dub-2A enzyme (Fig. 2). This was demonstrated by both mutations which substituted Asp323 with Ala323, and Asp323 with Asn323. This is surprising since this result argues the model suggested by Hu et al. [15]. Therefore, the detailed analysis of their molecular structure of Dub-2A may be helpful for investigating the mechanism of enzyme action and for isolating its substrates in vivo.