No abstract

Vascular development in mice only requires kinase-independent functions of FAK until E13.5, but kinase activity is needed for embryogenesis to complete.

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...

Autophagy is an evolutionarily conserved cellular process controlled through a set of essential autophagy genes (Atgs). However, there is increasing evidence that most, if not all, Atgs also possess functions independent of their requirement in canonical autophagy, making it difficult to distinguish the contributions of autophagy-dependent or -independent functions of a particular Atg to various biological processes. To distinguish these functions for FIP200 (FAK family-interacting protein of 200 kDa), an Atg in autophagy induction, we examined FIP200 interaction with its autophagy partner, Atg13. We found that residues 582-585 (LQFL) in FIP200 are required for interaction with Atg13, and mutation of these residues to AAAA (designated the FIP200-4A mutant) abolished its canonical autophagy function in vitro. Furthermore, we created a FIP200-4A mutant knock-in mouse model and found that specifically blocking FIP200 interaction with Atg13 abolishes autophagy in vivo, providing direct support for the essential role of the ULK1/Atg13/FIP200/Atg101 complex in the process beyond previous studies relying on the complete knockout of individual components. Analysis of the new mouse model showed that nonautophagic functions of FIP200 are sufficient to fully support embryogenesis by maintaining a protective role in TNFα-induced apoptosis. However, FIP200-mediated canonical autophagy is required to support neonatal survival and tumor cell growth. These studies provide the first genetic evidence linking an Atg's autophagy and nonautophagic functions to different biological processes in vivo.

Summary Angiosarcoma/lymphangiosarcoma is a rare malignancy with poor prognosis. We generated a mouse model with inducible endothelial cell-specific deletion of Tsc1 to examine mTORC1 signaling in lymphangiosarcoma. Tsc1 loss increased retinal angiogenesis in neonates, and led to endothelial proliferative lesions from vascular malformations to vascular tumors in adult mice. Sustained mTORC1 signaling was required for lymphangiosarcoma development and maintenance. Increased VEGF expression in tumor cells was seen, and blocking autocrine VEGF signaling abolished vascular tumor development and growth. We also found significant correlations between mTORC1 activation and VEGF, HIF1α, and c-Myc expression in human angiosarcoma samples. These studies demonstrated critical mechanisms of aberrant mTORC1 activation in lymphangiosarcoma, and validate the mice as a valuable model for further study.

No abstract

Background: FAK has both kinase and scaffolding functions. Results: Disruption of the function of FAK scaffolding to mediate endophilin A2 phosphorylation inhibits mammary tumor growth and metastasis in vivo by decreasing tumor cell markers for EMT and their MaCSCs activities. Conclusion:The function of FAK scaffolding is important for promoting mammary tumor progression. Significance: Targeting the scaffolding function of FAK may be important in breast cancer therapy.