Transforming growth factor-␤-activated kinase 1 (TAK1) plays an essential role in the tumor necrosis factor ␣ (TNF␣)-and interleukin-1␤ (IL-1␤)-induced IB kinase (IKK)/nuclear factor-B (NF-B) and c-Jun N-terminal kinase (JNK)/Tumor necrosis factor ␣ (TNF␣) 3 and interleukin-1␤ (IL-1␤) are two potent proinflammatory cytokines that play important roles in the regulation of immunity, inflammation, cell proliferation, differentiation, and apoptosis (1, 2). Cellular responses to TNF␣ and IL-1␤ are mediated by intracellular signaling pathways that control the activation of nuclear factor-B (NF-B) and activator protein 1 (AP-1) (3, 4).Upon binding to its receptor, TNF␣ induces formation of a receptor-associated complex, including the adaptor proteins TRADD, TRAF2, TRAF5, and RIP1, which subsequently leads to Lys 63 -linked polyubiquitination of TRAF2 and RIP1 (5-7). In contrast, IL-1␤ binding to its receptor induces a receptor-associated complex formation, including MyD88, IRAK1, IRAK4, and TRAF6, which is followed by Lys 63 -linked polyubiquitination of TRAF6 and IRAKs (8 -12). The formation of TRAF2-RIP1 and TRAF6-IRAK4 complexes as well as the Lys 63 -linked polyubiquitination of RIP1 and TRAF6 appear to enable the recruitment and activation of transforming growth factor-␤-activated kinase 1 (TAK1) through binding of the TAK1 regulatory subunits TAB2 and TAB3 to the Lys 63 -polyubiquitinated RIP1 and TRAF6. The activated TAK1 then triggers the activation of the IB kinase (IKK), c-Jun N-terminal kinase (JNK), and p38 MAPK (8,(13)(14)(15)(16)(17), which leads to activation of transcription factors NF-B and AP-1 and up-regulation of many genes encoding proinflammatory cytokines, chemokines, adhesion molecules, and proteolytic enzymes (18).The IKK complex consists of three subunits: two catalytic subunits, IKK␣ and IKK␤, and an essential regulatory subunit, IKK␥/NF-B essential modulator (NEMO) (3,19). Genetic studies have implicated that IKK␤ and IKK␥/NEMO are essential for the TNF␣-and IL-1␤-mediated . Phosphorylation of serine 177 and 181 residues in the activation loop is required for IKK␤ activation (23). IKK␥/NEMO has been indicated to bind Lys 63 -linked polyubiquitin chains (7,16,24,25). It is proposed that Lys 63 -polyubiquitin chains act as a scaffold to allow for assembly of a signaling complex that leads to IKK␤ activation. Once activated, IKK␤ phosphorylates IB proteins and leads to IB polyubiquitination with a Lys 48 -linked ubiquitin chain. Polyubiquitination-mediated degradation of IBs allows NF-B to translocate into the nucleus and activate NF-B-dependent gene expression (26).JNKs are members of three related mitogen-activated protein kinases (MAPKs), including the extracellular signal-regulated kinases (ERKs), JNKs, and p38 MAPKs (4, 27). JNKs and p38 MAPKs are involved in transmitting intracellular signals in

Lys63-linked polyubiquitination of TAK1 plays an important role in TNFα-induced NF-κB activation. Using a functional genomic approach, we have identified Ubiquitin Specific Peptidase 4 (USP4) as a deubiquitinase for TAK1. USP4 deubiquitinates TAK1 in vitro and in vivo. TNFα induces association of USP4 with TAK1 to deubiquitinate TAK1 and downregulate TAK1-mediated NF-κB activation. Overexpression of USP4 wild-type, but not deuibiquitinase-deficient C311A mutant, inhibits both TNFα- and TAK1/TAB1 co-overexpression-induced TAK1 polyubiquitination and NF-κB activation. Notably, knockdown of USP4 in HeLa cells enhances TNFα-induced TAK1 polyubiquitination, IKK phosphorylation, IκBα phosphorylation and ubiquitination as well as NF-κB-dependent gene expression. Moreover, USP4 negatively regulates IL-1β-, LPS-and TGFβ-induced NF-κB activation. Together, our results demonstrate that USP4 serves as a critical control to downregulate TNFα-induced NF-κB activation through deubiquitinating TAK1.

Ubiquitination and deubiquitination of receptor-interacting protein 1 (RIP1) play an important role in the positive and negative regulation of the tumor necrosis factor ␣ (TNF␣)-induced nuclear factor B (NF-B) activation. Using a combination of functional genomic and proteomic approaches, we have identified ubiquitin-specific peptidase 21 (USP21) as a deubiquitinase for RIP1. USP21 is constitutively associated with RIP1 and deubiquitinates RIP1 in vitro and in vivo. Notably, knockdown of USP21 in HeLa cells enhances TNF␣-induced RIP1 ubiquitination, IB kinase ␤ (IKK␤), and NF-B phosphorylation, inhibitor of NF-B ␣ (IB␣) phosphorylation and ubiquitination, as well as NF-B-dependent gene expression. Therefore, our results demonstrate that USP21 plays an important role in the down-regulation of TNF␣-induced NF-B activation through deubiquitinating RIP1. Transcription factor nuclear factor B (NF-B)3 plays an important role in controlling the expression of survival factors, cytokines, and proinflammatory molecules in a broad range of cellular responses (1-3). NF-B is sequestered in the cytoplasm by a family of inhibitory proteins called inhibitor of NF-B (IB) proteins in inactivated cells. Many intercellular stimuli are capable of triggering the activation of a signal transduction pathway that leads to the degradation of IB proteins through the 26 S proteasome (4 -6). Degradation of the IB proteins allows NF-B translocation from cytoplasm to the nucleus and activates the expression of the target genes (7).Upon binding of tumor necrosis factor ␣ (TNF␣), TNF receptor 1 (TNFR1) recruits several adaptor proteins, including receptorinteracting protein 1 (RIP1/RIPK1) and TNF receptor-associated factor 2 (TRAF2), to form a complex (8, 9). This TNFR1-associated complex initiates the activation of IB kinase (IKK), which phosphorylates IB protein and activates NF-B (10 -17).Protein ubiquitination is a crucial regulatory mechanism in various cellular processes, including cell cycle progression, the DNA damage response, and immune responses (18 -20). In the TNF␣-induced NF-B signal transduction pathway, the Lys 63 -linked polyubiquitination of RIP1 protein mediated by TRAF2 E3 ligase is essential for TNF␣-induced IKK/NF-B activation, whereas phosphorylation of the IB proteins by activated IKK leads to their Lys 48 -linked polyubiquitination, which labels it for its degradation by the 26 S proteasome (21).Several deubiquitinating enzymes, including CYLD, A20, Cezanne, ubiquitin-specific peptidase 15 (USP15), and USP31, have been suggested to be involved in the down-regulation of TNF␣-induced NF-B activation (22-26). However, it remains unclear how deubiquitination plays a role in the down-regulation of TNF␣-induced NF-B activation.The USPs belong to a subclass of the protein-deubiquitinating enzyme (DUB) superfamily that are categorized into five subclasses based on their ubiquitin-protease domains in the human genome and have been shown to be involved in a broad range of biological activities (27). Even though the USP subclass o...