Activation of transcription factor NF-kappa B by the adenovirus E3/19K protein requires its ER retention

We have recently shown that the accumulation of diverse viral and cellular membrane proteins in the ER activates the higher eukaryotic transcription factor NF-kappa B. 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-kappa B pathway is not known. Here we used the adenovirus early region protein E3/19K as a model to investigate the nature of the NF-kappa B-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-kappa B transcription factor. The activated NF-kappa B complexes contained p50/p65 and p50/c-rel heterodimers. E3/19K interaction with MHC class I was not important for NF-kappa B activation since mutant proteins which no longer bind MHC molecules remained fully capable of inducing NF-kappa B. However, activation of both NF-kappa B DNA binding and kappa B-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-kappa B-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-kappa B activation but not TNF-stimulated NF-kappa B induction can be inhibited by the intracellular Ca2+ chelator TMB-8. Moreover, treatment of cells with two inhibitors of the ER-resident Ca2+-dependent ATPase, thapsigargin and cyclopiazonic acid, which causes a rapid release of Ca2+ from the ER, strongly activated NF-kappa B. We therefore propose that ER overload activates NF-kappa B by causing Ca2+ release from the ER. Because NF-kappa B plays a key role in mounting an immune response, ER overload caused by viral proteins may constitute a simple antiviral response with broad specificity.