ATF6 ubiquitylation is required for its transcriptional activity and degradation

ATF6α is an endoplasmic reticulum (ER) transmembrane protein that senses the accumulation of misfolded proteins in the ER of cells during ER stress. Upon ER stress, ATF6α is proteolytically cleaved. The resulting N‐terminal fragment becomes an active transcription factor, i.e. N‐ATF6α, which induces genes that adaptively reprogram ER protein folding. N‐ATF6α exerts potent but transient transcriptional activation of its target genes; the transient nature of its activity is due in part to its rapid degradation upon engagement in transcription. The transcriptional activation domain (TAD) and degradation domain (degron) of N‐ATF6α map to the same N‐terminal region of N‐ATF6α, implying that transcriptional activity and degradation are linked. Moreover, mutations introduced into the TAD of N‐ATF6α that decrease its activity also decrease its degradation, indicating that N‐ATF6α degradation is coupled with its transcriptional activity. To elucidate how and why N‐ATF6α degradation and activation are coupled, we examined the mechanism of N‐ATF6α degradation, positing that ubiquitylation may target it for proteasome‐mediated degradation. Accordingly, this study focused primarily on delineating whether N‐ATF6α is ubiquitylated, and if so, what domain(s) of N‐ATF6α are ubiquitylated, as well as investigating the functional consequences of such ubiquitylation. Methods HeLa cells were co‐transfected with DNA constructs encoding FLAG‐tagged N‐ATF6α and with constructs encoding HA‐ubiquitin. To examine ubiquitylation, FLAG‐N‐ATF6α was FLAG immunoprecipitated (IP'd), then subjected to SDS‐PAGE followed by immunoblotting (IB) with anti‐HA or anti‐FLAG antibody. Specific ubiquitylation, i.e. the level of ubiquitylation per quantity of ATF6, was defined as the ratio of HA/FLAG. A series of mutations was introduced into N‐ATF6α to map the ubiquitylation domain(s) on N‐ATF6α and to assess the functional effects of the mutations that exhibited altered ubiquitylation. Transfected N‐ATF6α function was determined by assessing its ability to induce canonical ATF6 target genes by IB. Results In the absence of ER stress, wild type (WT) N‐ATF6α was ubiquitylated in a proteasome‐dependent manner in HeLa cells. A series of mutations in the N‐terminus of N‐ATF6α that progressively decreased its ability to induce ATF6 target genes coordinately decreased its specific ubiquitylation, as well as its degradation. Using this mutation approach, the ubiquitylation domain of N‐ATF6α was mapped to the N‐terminal 39 amino acids. To determine whether engagement in transcription affected ATF6 ubiquitylation, the effect of mutating the DNA binding domain (DBD) of WT N‐ATF6α was examined. Interestingly, the DBD mutation disengaged ATF6 from DNA, which led to a loss of its transcriptional activation, as expected, and to a dramatic increased its specific ubiquitylation. Conclusion N‐ATF6α is ubiquitylated in a domain within the first 39 amino acids of the N‐terminus. This ubiquitylation is required for full ATF6 activity as a transcription factor, as well as its degradation. Moreover, deubiquitylation, degradation and transcriptional engagement were shown to be coordinate processes. Support or Funding Information National Institutes of Health (NIH) grants R01 HL75573, R01 HL104535, R01 HL127439, and P01 HL085577 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .