Inhibitor of Bruton's Tyrosine Kinase‐α (IBTKα) in the Unfolded Protein Response

Abstract

Disruption of protein and membrane homeostasis in the endoplasmic reticulum (ER) occurs in response to diverse environmental and physiological stresses. The ensuing unfolded protein response (UPR) serves to alleviate stress damage by lowering the influx of nascent proteins into the stressed organelle concurrent with enhanced expression of stress adaptive proteins that serve to expand the ER and restore homeostasis. Phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2) is central to the translational and transcription regulation of the UPR. While eIF2α phosphorylation serves to dampen global translation during ER stress, selected mRNAs are preferentially translated. The selective translation is facilitated via specialized upstream open reading frames (uORFs) in the 5’ leaders of these mRNAs. Our laboratory previously showed that there is a significant increase in the expression of IBTKα mRNA and protein in response to ER stress. IBTKα is a large multi-domain protein that engages with CUL3 and is suggested to ubiquitylate proteins involved in the regulation of protein homeostasis. The 5’ leader of human IBTKα mRNA contains 4 uORFs, and uORF1 and uORF2 are phylogenetically conserved. To determine the translational regulation of IBTKα, we used a translational reporter expressing the 5’ leader of IBTKα mRNA fused to firefly luciferase coding sequence. Mutation of the uORFs start codon from ATG to AGG was made to individually disrupt their functions. Wild type and mutant versions of IBTKα vectors were transiently transfected in HEK293T cells and these cells were treated with a pharmacological inducer of ER stress, thapsigargin, or vehicle, and luciferase activity was determined as a measure of translation control. We determined that uORF2 serves as a major inhibitory uORF that can be selectively bypassed during ER stress to enhance translation of the IBTKα coding sequence. Mutational analyses of conserved RNA stem-loop structures downstream of uORF2 enhances IBTKα translation in a fashion suggested to be linked with reinitiation following the inhibitory uORF2. Our study provides an example of how the secondary RNA structure of specific mRNA can contribute to its regulation along with uORFs present in the 5’ leader of mRNA in the UPR. For IBTKα functional studies, we are utilizing ubiquitin enrichment analyses to determine downstream targets of the IBTKα-CUL3 complex in the UPR. Finally, we are generating a knockout of IBTKα in mice to further characterize the function of IBTKα in ER stress. Our studies will provide insights into the regulation and function of a new UPR member, IBTKα, in the UPR.