title: Effects of the isoform-specific characteristics of ATF6 alpha and ATF6 beta on endoplasmic reticulum stress response gene expression and cell viability.
abstract: The endoplasmic reticulum (ER)-transmembrane proteins, ATF6 alpha and ATF6 beta, are cleaved during the ER stress response (ERSR). The resulting N-terminal fragments (N-ATF6 alpha and N-ATF6 beta) have conserved DNA-binding domains and divergent transcriptional activation domains. N-ATF6 alpha and N-ATF6 beta translocate to the nucleus, bind to specific regulatory elements, and influence expression of ERSR genes, such as glucose-regulated protein 78 (GRP78), that contribute to resolving the ERSR, thus, enhancing cell viability. We previously showed that N-ATF6 alpha is a rapidly degraded, strong transcriptional activator, whereas beta is a slowly degraded, weak activator. In this study we explored the molecular basis and functional impact of these isoform-specific characteristics in HeLa cells. Mutants in the transcriptional activation domain or DNA-binding domain of N-ATF6 alpha exhibited loss of function and increased expression, the latter of which suggested decreased rates of degradation. Fusing N-ATF6 alpha to the mutant estrogen receptor generated N-ATF6 alpha-MER, which, without tamoxifen exhibited loss-of-function and high expression, but in the presence of tamoxifen N-ATF6 alpha-MER exhibited gain-of-function and low expression. N-ATF6 beta conferred loss-of-function and high expression to N-ATF6 alpha, suggesting that ATF6 beta is an endogenous inhibitor of ATF6 alpha. In vitro DNA binding experiments showed that recombinant N-ATF6 beta inhibited the binding of recombinant N-ATF6 alpha to an ERSR element from the GRP78 promoter. Moreover, siRNA-mediated knock-down of endogenous ATF6 beta increased GRP78 promoter activity and GRP78 gene expression, as well as augmenting cell viability. Thus, the relative levels of ATF6 alpha and -beta, may contribute to regulating the strength and duration of ATF6-dependent ERSR gene induction and cell viability.
authors.affiliation: San Diego State University Heart Institute and Department of Biology, San Diego State University, San Diego, California 92182, USA.
authors.forename: Donna J
authors.initials: DJ
authors.lastname: Thuerauf
authors.forename: Marie
authors.initials: M
authors.lastname: Marcinko
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Belmont
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Glembotski
mesh_terms: D051702:Activating Transcription Factor 6 / Q000737:chemistry*; D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry*; D002470:Cell Survival; D003587:Cytomegalovirus / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D015533:Transcriptional Activation; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: C497111:ATF6 protein, human; C487807:ATF6B protein, human; D051702:Activating Transcription Factor 6; D050976:Basic-Leucine Zipper Transcription Factors; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D004247:DNA; D001616:beta-Galactosidase