The GCN2-ATF4 signaling axis orchestrates the hepatic transcriptional stress response to short-term sulfur amino acid deprivation.

Mammals rely on the integrated stress response (ISR) to maintain essential amino acid (EAA) homeostasis. The kinase GCN2 is a key ISR sensor that is rapidly activated by uncharged tRNAs during EAA deprivation, leading to eIF2α phosphorylation and selective translation of ATF4. ATF4 subsequently orchestrates a transcriptional program regulating amino acid metabolism, redox balance, and autophagy. In this study, we investigated the role of GCN2 in the early hepatic transcriptional response to dietary sulfur amino acids (SAA; methionine and cysteine) deprivation. Using ATF4-luciferase reporter mice, we demonstrate that short-term SAA deprivation rapidly activates the eIF2α-ATF4 pathway within 3 hours, with activation primarily localized to the liver. Complementary in vivo and ex vivo approaches revealed that genetic deletion or pharmacological inhibition of GCN2 abolishes early eIF2α phosphorylation and induction of ATF4 target gene, while PERK is dispensable for this response. Furthermore, GCN2 controls the induction of multiple adaptive transcriptional programs involved in amino acid transport, aminoacyl-tRNA synthesis, autophagy, serine biosynthesis, one-carbon metabolism and glutathione degradation highlighting a coordinated adaptive response to acute SAA deprivation. These findings establish GCN2 as a major sensor mediating the early hepatic response to SAA deprivation, and define a transcriptional program essential for maintaining amino acid homeostasis. In contrast, Fgf21 induction occurs independently of GCN2, indicating the existence of parallel adaptive mechanisms. Collectively, this work provides new insight into the early dynamics and molecular specificity of ISR activation in response to acute dietary SAA deprivation.