Protein ubiquitination is an essential posttranslational modification that is implicated in many biological processes (14
). Ubiquitin is a small protein composed of 76 amino acids. It contains 7 lysine residues (K6, K11, K27, K29, K33, K48, and K63). Multiple ubiquitin monomers can become covalently linked, and polyubiquitin molecules linked through the lysine 48 residue (K48) are known to modulate protein degradation. In contrast, polyubiquitin molecules linked through the lysine 63 (K63) residue do not induce degradation but influence protein localization, protein-protein interaction, protein functional activation, and other activities (14
). The addition of ubiquitin to or the removal of ubiquitin from protein substrates can reversibly and dynamically change protein functions, and these reactions are executed by ubiquitin ligases and deubiquitinases (44
). Accordingly, the ubiquitination process is mediated by the serial actions of E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase (14
), and deubiquitination is through deubiquitining enzymes (DUBs) that directly remove ubiquitin molecules from their substrates (32
). Based on sequence analyses, approximately 90 DUB genes have been identified in the human genome. These DUBs are divided into 5 subclasses according to their protein sequences: the ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs), Machado-Joseph disease protein domain proteases (MJDs), ovarian tumor proteases (OTUs), and JAMM motif proteases (32
). The specificities and actions of these various deubiquitinases remain to be fully characterized.
In mammals, NF-κB signaling plays key roles in inflammation, cell proliferation, and apoptosis (36
). Like phosphorylation, ubiquitination is an important posttranslational modification that can regulate NF-κB activity (43
). For example, the ubiquitination of a regulatory subunit in the IKK complex, IKKγ which is also known as NEMO, has a central role in signal transduction. It has been shown that K63-linked linear conjugation of ubiquitin to IKKγ positively regulates NF-κB signaling (47
). In addition, the ubiquitination of tumor necrosis factor receptor-associated factors (TRAFs) is also important for IKK activation. TRAF6 is an E3 ubiquitin ligase, and it performs self-ubiquitination through K63-linked chains upon cellular activation through Toll-like receptors (TLRs) and cytokine receptors (8
). Moreover, in the canonical NF-κB pathway, IκBα, which sequesters NF-κB proteins in the cytoplasm in an inactive state, is conjugated with K48-linked polyubiquitin chains and is proteasomally degraded when cells are stimulated to activate NF-κB (4
). Similarly, cellular activation induces the ubiquitination and proteosomal processing of NF-κB2 p100 to p52, allowing NF-κB RelB/p52 dimers to translocate into the nucleus.
The role of DUBs in the NF-κB pathway has also been studied. For example, the familial cylindromatosis tumor suppressor CYLD is one of the DUBs that have been found to suppress NF-κB activity. CYLD has been shown to bind IKKγ and to reduce the ubiquitination of TRAF2, TRAF6, and IKKγ (3
). A20 is a second well-studied DUB that negatively regulates NF-κB activation by reducing the ubiquitination of TRAF2, TRAF6, and RIP1 (17
). A20 has dual activities in ubiquitination and deubiquitination. Hence, A20 with TAX1BP1 as a cofactor promotes the cleavage of K63-linked polyubiquitin chains on RIP1, and A20 with E3 ligase Itch can conjugate K48-linked chains on RIP for proteosomal degradation (39
). Recently, it was also found that A20 can inhibit the ubiquitination of TRAF2 and TRAF6 by dissociating complexes composed of TRAFs and E2 ubiquitin-conjugating enzymes (41
Infection by human T cell leukemia virus type 1 (HTLV-1) causes a fatal hematopoietic malignancy, adult T cell leukemia (ATL) (29
), and one of its key regulatory proteins, Tax, plays important roles in viral pathogenesis (2
). Tax (29
) can potently activate NF-κB through both the canonical and noncanonical pathways (15
). Tax was recently found to inhibit A20 function by disrupting its interaction with TAX1BP1 and Itch (39
). Currently, how other DUBs may contribute to Tax-induced NF-κB signaling has not been studied. Here, we report on the characterization of USP20 for its regulation of Tax- and TRAF6-mediated activation of NF-κB.