Methods for analyzing eIF2 kinases and translational control in the unfolded protein response.
Endoplasmic reticulum (ER) stress induces a program of translational and transcriptional regulation, designated the unfolded protein response (UPR), that collectively remedies stress damage and restores ER homeostasis. The protein kinase PERK facilitates the translational control arm of the UPR by phosphorylation of eIF2, a translation initiation factor that combines with GTP to escort initiator Met-tRNA(i)(Met) to the ribosomal machinery during the initiation of protein synthesis. Phosphorylation of the alpha subunit of eIF2 on serine-51 inhibits global translation initiation, which reduces the influx of nascent polypeptides into the overloaded ER. eIF2 phosphorylation also facilitates the preferential translation of stress-related mRNAs, such as ATF4 which in turn activates the transcription of UPR genes. In this chapter, we present experimental strategies and methods for establishing and characterizing global and gene-specific translation control induced by eIF2 phosphorylation (eIF2α~P) during ER stress. These methods include assays for the detection of eIF2α~P and its target genes. We also discuss strategies to address whether a given ER stress condition triggers eIF2α~P through PERK, as opposed to other stress conditions activating alternative members of the eIF2 kinase family. Additionally, experimental descriptions are provided for detecting and quantifying a repression in global translation initiation, and identifying stress-induced preferential translation, such as that described for ATF4. Together, these experimental descriptions will provide a useful molecular "toolkit" to study each feature of the translational control processes invoked during ER stress.
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
Brian F
BF
Teske
Thomas D
TD
Baird
Ronald C
RC
Wek
D051701:Activating Transcription Factor 4 / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001681:Biological Assay / Q000295:instrumentation / Q000379:methods*; D003545:Cysteine / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000502:physiology; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D013312:Stress, Physiological; D014158:Transcription, Genetic; D056811:Unfolded Protein Response / Q000502:physiology*; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism
D051701:Activating Transcription Factor 4; D003545:Cysteine; D015852:Eukaryotic Initiation Factor-2; D008715:Methionine; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase