Proteasome activity is frequently enhanced in cancer to accelerate metastasis and tumorigenesis. REGγ, a proteasome activator known to promote p53/p21/p16 degradation, is often overexpressed in cancer cells. Here we show that p53/TGF-β signalling inhibits the REGγ–20S proteasome pathway by repressing REGγ expression. Smad3 and p53 interact on the REGγ promoter via the p53RE/SBE region. Conversely, mutant p53 binds to the REGγ promoter and recruits p300. Importantly, mutant p53 prevents Smad3/N-CoR complex formation on the REGγ promoter, which enhances the activity of the REGγ–20S proteasome pathway and contributes to mutant p53 gain of function. Depletion of REGγ alters the cellular response to p53/TGF-β signalling in drug resistance, proliferation, cell cycle progression and proteasome activity. Moreover, p53 mutations show a positive correlation with REGγ expression in cancer samples. These findings suggest that targeting REGγ–20S proteasome for cancer therapy may be applicable to human tumours with abnormal p53/Smad protein status. Furthermore, this study demonstrates a link between p53/TGF-β signalling and the REGγ–20S proteasome pathway, and provides insight into the REGγ/p53 feedback loop.
As a latent transcription factor, nuclear factor κB (NF-κB) translocates from the cytoplasm into the nucleus upon stimulation and mediates the expression of genes that are important in immunity, inflammation, and development. However, little is known about how it is regulated inside the nucleus. By a two-hybrid approach, we identify a prefoldin-like protein, ubiquitously expressed transcript (UXT), that is expressed predominantly and interacts specifically with NF-κB inside the nucleus. RNA interference knockdown of UXT leads to impaired NF-κB activity and dramatically attenuates the expression of NF-κB–dependent genes. This interference also sensitizes cells to apoptosis by tumor necrosis factor-α. Furthermore, UXT forms a dynamic complex with NF-κB and is recruited to the NF-κB enhanceosome upon stimulation. Interestingly, the UXT protein level correlates with constitutive NF-κB activity in human prostate cancer cell lines. The presence of NF-κB within the nucleus of stimulated or constitutively active cells is considerably diminished with decreased endogenous UXT levels. Our results reveal that UXT is an integral component of the NF-κB enhanceosome and is essential for its nuclear function, which uncovers a new mechanism of NF-κB regulation.
Interferon regulatory factor 3 (IRF3) plays a crucial role in mediating cellular responses to virus intrusion. The protein kinase TBK1 is a key regulator inducing phosphorylation of IRF3. The regulatory mechanisms during IRF3 activation remain poorly characterized. In the present study, we have identified by yeast two-hybrid approach a specific interaction between IRF3 and chaperone heat-shock protein of 90 kDa (Hsp90). The C-terminal truncation mutant of Hsp90 is a strong dominant-negative inhibitor of IRF3 activation. Knockdown of endogenous Hsp90 by RNA interference attenuates IRF3 activation and its target gene expressions. Alternatively, Hsp90-specific inhibitor geldanamycin (GA) dramatically reduces expression of IRF3-regulated interferon-stimulated genes and abolishes the cytoplasm-to-nucleus translocation and DNA binding activity of IRF3 in Sendai virus-infected cells. Significantly, virus-induced IRF3 phosphorylation is blocked by GA, whereas GA does not affect the protein level of IRF3. In addition, TBK1 is found to be a client protein of Hsp90 in vivo. Treatment of 293 cells with GA interferes with the interaction of TBK1 and Hsp90, resulting in TBK1 destabilization and its subsequent proteasome-mediated degradation. Besides maintaining stability of TBK1, Hsp90 also forms a novel complex with TBK1 and IRF3, which brings TBK1 and IRF3 dynamically into proximity and facilitates signal transduction from TBK1 to IRF3. Our study uncovers an essential role of Hsp90 in the virus-induced activation of IRF3. INTRODUCTIONToll-like receptors (TLRs) played a crucial role in innate immunity by recognizing structurally conserved bacterial and viral components termed pathogen-associated molecular patterns (PAMPs) (Medzhitov and Janeway, 1998). Eleven TLRs had been cloned in mammals, and each receptor had been reported to recognize a unique set of PAMPs (Akira and Takeda, 2004). Many studies have shown that TLR3 mediated the response to the viral-associated PAMPs (e.g., the double-stranded RNA [dsRNA]), whereas TLR4 recognized the bacterial-associated components, including lipopolysaccharide (LPS) and Gram-positive lipoteichoic acids (Takeuchi et al., 1999;Alexopoulou et al., 2001;Takeuchi and Akira, 2001). On stimulation by corresponding PAMPs, both TLR3 and TLR4 had been shown to activate interferon regulatory factor 3 (IRF3) through the MyD88-independent pathway (Akira and Takeda, 2004;Boehme and Compton, 2004;Palsson-McDermott and O'Neill, 2004;Bowie and Haga, 2005).IRF3, originally identified in a variety of tissues based on homology with other known IRF family members, was expressed constitutively without activity in the cytoplasm; and no change in the relative levels of IRF3 mRNA was observed in virus-infected cells (Au et al., 1995). Recent investigations found that IRF3 was an important transcriptional regulator of antiviral immune response, mediating the expression of type I IFN and other Interferon stimulated genes (ISGs) (Schafer et al., 1998;Doyle et al., 2002;Elco et al., 2005). After LPS stimulation or...
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