An In Vitro System to Study Mammalian GCN2 Regulation

General control non-derepressible 2 (GCN2) is an eIF2α kinase responsible for eliciting the integrated stress response (ISR) under amino acid starvation conditions. How GCN2 can sense amino acid starvation and become active in mammalian systems is not fully understood as there are a plethora of protein cofactors and post-translational modifications which may facilitate this transition. In this chapter, we describe the purification of recombinantly expressed human GCN2 from an insect cell expression system, and two assays which can quantify its kinase activity. Firstly, we describe an Western blot assay which can be used to directly assess phosphorylation of a downstream target, eIF2α. Secondly, we describe a luciferase-coupled kinase assay using an arginine-serine repeat peptide of eIF2α as the substrate. Together, these assays allow for the determination of GCN2 activity under a variety of conditions.