title: Activation of transcription factor NF-kappaB by the adenovirus E3/19K protein requires its ER retention.
abstract: We have recently shown that the accumulation of diverse viral and cellular membrane proteins in the ER activates the higher eukaryotic transcription factor NF-kappaB. This defined a novel ER-nuclear signal transduction pathway, which is distinct from the previously described unfolded protein response (UPR). The well characterized UPR pathway is activated by the presence of un- or malfolded proteins in the ER. In contrast, the ER stress signal which activates the NF-kappaB pathway is not known. Here we used the adenovirus early region protein E3/19K as a model to investigate the nature of the NF-kappaB-activating signal emitted by the ER. E3/19K resides in the endoplasmic reticulum where it binds to MHC class I molecules, thereby preventing their transport to the cell surface. It is maintained in the ER by a retention signal sequence in its carboxy terminus, which causes the protein to be continuously retrieved to the ER from post-ER compartments. Mutation of this sequence allows E3/19K to reach the cell surface. We show here that expression of E3/19K potently activates a functional NF-kappaB transcription factor. The activated NF-kappaB complexes contained p50/p65 and p50/c-rel heterodimers. E3/19K interaction with MHC class I was not important for NF-kappaB activation since mutant proteins which no longer bind MHC molecules remained fully capable of inducing NF-kappaB. However, activation of both NF-kappaB DNA binding and kappaB-dependent transactivation relied on E3/19K ER retention: mutants, which were expressed on the cell surface, could no longer activate the transcription factor. This identifies the NF-kappaB-activating signal as the accumulation of proteins in the ER membrane, a condition we have termed "ER overload." We show that ER overload-mediated NF-kappaB activation but not TNF-stimulated NF-kappaB induction can be inhibited by the intracellular Ca2+ chelator TMB-8. Moreover, treatment of cells with two inhibitors of the ER-resident Ca(2+) -dependent ATPase, thapsigargin and cyclopiazonic acid, which causes a rapid release of Ca2+ from the ER, strongly activated NF-kappaB. We therefore propose that ER overload activates NF-kappaB by causing Ca2+ release from the ER. Because NF-kappaB plays a key role in mounting an immune response, ER overload caused by viral proteins may constitute a simple antiviral response with broad specificity.
authors.affiliation: Institute of Biochemistry, Albert-Ludwigs-University, Freiburg, Germany.
authors.forename: H L
authors.initials: HL
authors.lastname: Pahl
authors.forename: M
authors.initials: M
authors.lastname: Sester
authors.forename: H G
authors.initials: HG
authors.lastname: Burgert
authors.forename: P A
authors.initials: PA
authors.lastname: Baeuerle
mesh_terms: D017503:Adenovirus E3 Proteins / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism*; D015398:Signal Transduction; D015533:Transcriptional Activation
chemical_list: D017503:Adenovirus E3 Proteins; D016328:NF-kappa B