| pmid | 10848577 | |||||
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| title | Stat1 as a component of tumor necrosis factor alpha receptor 1-TRADD signaling complex to inhibit NF-kappaB activation. | |||||
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| journal | Mol Cell Biol | |||||
| year | 2000 | |||||
| full_text_available | true | |||||
| full_text_extraction_method | html | |||||
| pmcid | PMC85828 | |||||
| doi | 10.1128/MCB.20.13.4505-4512.2000 |
Stat1 as a component of tumor necrosis factor alpha receptor 1-TRADD signaling complex to inhibit NF-kappaB activation.
Authors: Wang Y, Wu TR, Cai S, Welte T, Chin YE Journal: Mol Cell Biol (2000) DOI: 10.1128/MCB.20.13.4505-4512.2000 PMC: PMC85828
- Mol Cell Biol. 2000 Jul;20(13):4505-12. doi: 10.1128/MCB.20.13.4505-4512.2000.
Stat1 as a component of tumor necrosis factor alpha receptor 1-TRADD signaling complex to inhibit NF-kappaB activation.
Wang Y(1), Wu TR, Cai S, Welte T, Chin YE.
Author information: (1)Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA.
Activated tumor necrosis factor alpha (TNF-alpha) receptor 1 (TNFR1) recruits TNFR1-associated death domain protein (TRADD), which in turn triggers two opposite signaling pathways leading to caspase activation for apoptosis induction and NF-kappaB activation for antiapoptosis gene upregulation. Here we show that Stat1 is involved in the TNFR1-TRADD signaling complex, as determined by employing a novel antibody array screening method. In HeLa cells, Stat1 was associated with TNFR1 and this association was increased with TNF-alpha treatment. TNFR1 signaling factors TRADD and Fas-associated death domain protein (FADD) were also found to interact with Stat1 in a TNF-alpha-dependent process. Our in vitro recombinant protein-protein interaction studies demonstrated that Stat1 could directly interact with TNFR1 and TRADD but not with FADD. Interaction between Stat1 and receptor-interacting protein (RIP) or TNFR-associated factor 2 (TRAF2) was not detected. Examination of Stat1-deficient cells showed an apparent increase in TNF-alpha-induced TRADD-RIP and TRADD-TRAF2 complex formation, while interaction between TRADD and FADD was unaffected. As a consequence, TNF-alpha-mediated I-kappaB degradation and NF-kappaB activation were markedly enhanced in Stat1-deficient cells, whereas overexpression of Stat1 in 293T cells blocked NF-kappaB activation by TNF-alpha. Thus, Stat1 acts as a TNFR1-signaling molecule to suppress NF-kappaB activation.
DOI: 10.1128/MCB.20.13.4505-4512.2000 PMCID: PMC85828 PMID: 10848577 [Indexed for MEDLINE]
Abstract
Activated tumor necrosis factor alpha (TNF-α) receptor 1 (TNFR1) recruits TNFR1-associated death domain protein (TRADD), which in turn triggers two opposite signaling pathways leading to caspase activation for apoptosis induction and NF-κB activation for antiapoptosis gene upregulation. Here we show that Stat1 is involved in the TNFR1-TRADD signaling complex, as determined by employing a novel antibody array screening method. In HeLa cells, Stat1 was associated with TNFR1 and this association was increased with TNF-α treatment. TNFR1 signaling factors TRADD and Fas-associated death domain protein (FADD) were also found to interact with Stat1 in a TNF-α-dependent process. Our in vitro recombinant protein-protein interaction studies demonstrated that Stat1 could directly interact with TNFR1 and TRADD but not with FADD. Interaction between Stat1 and receptor-interacting protein (RIP) or TNFR-associated factor 2 (TRAF2) was not detected. Examination of Stat1-deficient cells showed an apparent increase in TNF-α-induced TRADD-RIP and TRADD-TRAF2 complex formation, while interaction between TRADD and FADD was unaffected. As a consequence, TNF-α-mediated I-κB degradation and NF-κB activation were markedly enhanced in Stat1-deficient cells, whereas overexpression of Stat1 in 293T cells blocked NF-κB activation by TNF-α. Thus, Stat1 acts as a TNFR1-signaling molecule to suppress NF-κB activation.
DISCUSSION
The data presented here demonstrate that Stat1 recruitment by TNFR1 can block NF-κB activation, serving as an important mechanism for facilitating TNF-α-mediated apoptosis induction. Indeed, in some cells, the NF-κB pathway is inactive in response to TNF-α ( 26 ). TRADD can interact with a number of TNF-α signaling proteins, and the balance between the TRADD-FADD and TRADD-RIP or TRADD-TRAF2 signaling complexes decides whether cells live or die. Although TRADD independently interacted with Stat1, RIP, and TRAF2, coprecipitation of Stat1 with RIP or TRAF2 was not detected. This indicates that Stat1 competes with RIP and TRAF2 to bind TRADD. An enhanced TRAF2 expression level in Stat1-deficient cells can be responsible for enhanced NF-κB activation. Nevertheless, in the presence of Stat1, interaction between TRADD and TRAF2 or between TRADD and RIP was dramatically blocked. Stat1 may therefore affect NF-κB activation at two levels: (i) by blocking interaction between TRAF2 and TRADD and (ii) by blocking TRAF2 gene expression in cells. However, NF-κB activation by TNF-α was not impaired in TRAF2 knockout animals even though TRAF2 has been considered as one possible link to NF-κB activation ( 17 , 28 ). Therefore, NF-κB-dependent TRAF2 upregulation by TNF-α may be mainly involved in an NF-κB-independent antiapoptosis process. RIP is a serine/threonine kinase and has been considered as an important mediator of NF-κB activation by TRADD. In RIP-deficient cells, a failure of TNF-α to activate the transcription factor NF-κB was reported, suggesting that RIP-TRADD interaction plays a more important role in NF-κB activation by TNF-α ( 15 ). However, Stat1 facilitates TNF-α-mediated apoptosis induction at the TNFR1-TRADD complex formation-proximal step, either by displacing RIP and/or TRAF2 from TRADD or by disrupting other components required to execute the NF-κB activation signals (Fig. 7 ). Therefore, Stat1 should be a useful reagent for dissecting NF-κB activation initiated by other events, without excluding direct or indirect interaction between Stat1 and NF-κB ( 22 ). Although Stat1 and NF-κB can interact in nuclei with some nuclear factors, such as CBP for gene regulation, Stat1 does not translocate into nuclei for transcriptional activation in cells that have received TNF-α stimulation. Moreover, Stat1 suppresses NF-κB activity at a stage above I-κB degradation (Fig. 5 A). Therefore, it is unlikely that enhanced NF-κB activity in Stat1-deficient cells is due to the lack of competition between Stat1 and NF-κB for a common nuclear factor.
Stat1 deficiency on the one hand downregulates proapoptosis genes, such as caspase genes ( 5 , 16 ), and on the other hand enhances NF-κB activity leading to the upregulation of antiapoptosis genes, such as the TRAF2 gene. It therefore adds an additional mechanism underlying TNF-α-mediated protection. Although Stat1 undergoes tyrosine phosphorylation upon TNF-α stimulation, its DNA binding activity is extremely weak or undetectable. These results are in agreement with a previous study ( 8 ). Some growth factor or cytokine receptors, such as hepatocyte growth factor receptor, CD40, and angiotensin II receptor, have been reported to recruit and induce tyrosine phosphorylation of Stat proteins that exhibit poor or undetectable DNA binding activity ( 4 , 9 , 19 ). Thus, Stat proteins may primarily serve as signal transducers when recruited by these receptors. It has been recently reported that tyrosine-phosphorylated Stat proteins lose their DNA binding activity by binding to other protein factors, such as PIAS ( 6 ). Similarly, Stat1 did not translocate into nuclei upon TNF-α stimulation, probably due to its formation of a complex with TRADD in the cytoplasm. Stat proteins can be recruited by a variety of growth factor or cytokine receptors in a constitutive or stimulation-dependent manner ( 7 ). Although there is no apparent homology between TNFR1 and TNFR2 in their cytoplasmic domains, Stat1 coprecipitated with them both. It is currently unclear which motifs of these receptors are responsible for recruiting Stat.
The reduced interaction between Y701F-Stat1 and TRADD (Fig. 3 E) is consistent with the reduced effect of Y701F-Stat1 on TRADD-dependent NF-κB activation observed in Fig. 5 E. These results suggest that tyrosine phosphorylation at the Y701 residue of Stat1 may be necessary for Stat1 to interact with TRADD. Currently, it is unclear whether tyrosine phosphorylation at residues other than Y701 is involved in mediating protein-protein interaction. The role of Stat proteins as adapters mediating signal transduction has been previously observed. In alpha interferon receptor, for instance, Stat2 serves as the adapter for Stat1 activation and Stat3 functions to couple another signaling pathway, i.e., the PI 3-kinase, to the receptor ( 18 , 21 ). Our results provide evidence that tyrosine-phosphorylated Stat1 is involved in TNFR1 signal transduction. Rather than functioning as a nuclear transcription factor, Stat1 forms complexes with TRADD in the cytoplasm and regulates TRADD-mediated NF-κB activation.
The antibody array technology applied here allows for massive parallel protein analysis, a rapid and efficient method for large-scale protein-protein interaction examination. Antibody arrays can greatly increase the speed of experimental progress and will be valuable in the study of functional proteomics.