Abstract
Amino acids exert many biological functions, serving as allosteric regulators and neurotransmitters, as constituents in proteins and as nutrients. GCN2-mediated phosphorylation of eukaryotic initiation factor 2 alpha (elF2α) restores homeostasis in response to amino acid starvation (AAS) through the inhibition of the general translation and upregulation of amino acid biosynthetic enzymes and transporters by activating the translation of Gcn4 and ATF4 in yeast and mammals, respectively. GCN1 is a GCN2-binding protein that possesses an RWD binding domain (RWDBD) in its C-terminus. In yeast, Gcn1 is essential for Gcn2 activation by AAS; however, the roles of GCN1 in mammals need to be established. Here, we revealed a novel role of GCN1 that does not depend on AAS by generating two Gcn1 mutant mouse lines: Gcn1-knockout mice (Gcn1 KO mice (Gcn1-/-)) and RWDBD-deleted mutant mice (Gcn1ΔRWDBD mice). Both mutant mice showed growth retardation, which was not observed in the Gcn2 KO mice, such that the Gcn1 KO mice died at the intermediate stage of embryonic development because of severe growth retardation, while the Gcn1ΔRWDBD embryos showed mild growth retardation and died soon after birth, most likely due to respiratory failure. Extension of pregnancy by 24 h through the administration of progesterone to the pregnant mothers rescued the expression of differentiation markers in the lungs and prevented lethality of the Gcn1ΔRWDBD pups, indicating that perinatal lethality of the Gcn1ΔRWDBD embryos was due to simple growth retardation. Similar to the yeast Gcn2/Gcn1 system, AAS- or UV irradiation-induced elF2α phosphorylation was diminished in the Gcn1ΔRWDBD mouse embryonic fibroblasts (MEFs), suggesting that GCN1 RWDBD is responsible for GCN2 activity. In addition, we found reduced cell proliferation and G2/M arrest accompanying a decrease in Cdk1 and Cyclin B1 in the Gcn1ΔRWDBD MEFs. Our results demonstrated, for the first time, that GCN1 is essential for both GCN2-dependent stress response and GCN2-independent cell cycle regulation.
Highlights
Translational regulation through the phosphorylation of eukaryotic initiation factor 2 alpha at Ser51 is instigated by a wide range of stresses that regulate protein synthesis and generate cytoprotective responses and is called the integrated stress response (ISR) [1,2]
Gcn1 is required for Gcn2 activation by amino acid starvation (AAS), but the roles of GCN1 in mammals remain to be established
We show that GCN1 is involved in GCN2-mediated eIF2α phosphorylation after AAS and UV radiation by generating Gcn1 mutant mice
Summary
Translational regulation through the phosphorylation of eukaryotic initiation factor 2 alpha (elF2α) at Ser is instigated by a wide range of stresses that regulate protein synthesis and generate cytoprotective responses and is called the integrated stress response (ISR) [1,2]. ElF2α phosphorylation increases the translation of a range of mRNAs having an upstream open reading frame in the 5’-UTR, such as the transcription factor ATF4 [1,2]. Upon AAS, uncharged tRNA binds to the HisRS-like domain located at the C-terminus of Gcn and leads to its activation. Gcn bound to the ribosome presumably transfers the uncharged tRNA at the A-site of the ribosome to Gcn and is essential for Gcn activation by AAS [7]
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