GCN1 couples GCN2 to ribosomal state to initiate amino acid response pathway signaling.

During nutrient deprivation, activation of the protein kinase GCN2 regulates cell survival and metabolic homeostasis. In addition to amino acid stress, GCN2 is activated by a variety of cellular stresses. GCN2 activation has been linked to its association with uncharged tRNAs, specific ribosomal proteins, and conditions of translational arrest, but their relative contribution to activation is unclear. Here, we used in vitro translation to reconstitute GCN2 activation by amino acid stress and compared collided ribosome populations induced by diverse translational stressors. Initiation of GCN2 signaling required the di-ribosome sensor GCN1, which recruits GCN2 to ribosomes in a collision-dependent manner, where GCN2 becomes activated by key ribosomal interactions and stably associated with collided ribosomes. Our findings define the molecular requirements and dynamics of GCN2 activation. Editor's summary: When cells experience a lack of amino acids, they initiate a stress response signaled by activations of the protein kinase GCN2. Zhou et al. used an in vitro translation system to refine our understanding of how this activation of GCN2 occurs. In their experiments, the critical factor appeared to be the collision of ribosomes that occurs when aminoacyl-transfer RNAs are not available and a peptide chain cannot be elongated. The trailing ribosomes then collide with the stalled ribosome, and GCN2 was found to be associated with this molecular traffic jam. There, the presence of uncharged transfer RNAs, another consequence of a shortage of amino acids, also promoted the phosphorylation and activation of GCN2. —L. Bryan Ray INTRODUCTION: GCN2 is one of four mammalian kinases that sense different cellular stresses or pathological conditions to initiate the integrated stress response (ISR). This adaptive cellular response inhibits global protein synthesis while reprogramming cellular outputs to restore physiologic homeostasis. GCN2 is the only ISR kinase that is conserved throughout eukaryotes. A variety of cellular stresses, such as nucleic acid damage, can activate GCN2 but it is best characterized, along with its binding partner GCN1, as a key component of the amino acid response (AAR), the signaling arm of the ISR that detects amino acid insufficiency during protein synthesis. Despite extensive study, the mechanism of GCN2 activation within the complex milieu of stress response signaling remains unresolved. GCN2 activation has been variously attributed to its direct binding of uncharged tRNAs during amino acid stress, to its interaction with specific ribosomal proteins, and to conditions associated with translational arrest, such as ribosomal collisions. The relative importance of these distinct events, however, and how they relate to each other to activate GCN2, especially across different cellular responses, is unknown. A major limitation to resolving this question has been the absence of a biochemically tractable system that reconstitutes amino acid–induced GCN2 activation in the context of these other events. RATIONALE: The development of a cell-free in vitro translation system enabled us to follow the regulated activation of GCN2 at collided ribosomes in response to amino acid stress induction by the glutamyl-prolyl-tRNA synthetase inhibitor halofuginone (HF). This system allowed us to analyze both defined mRNA substrates and mutants of AAR pathway components, to uncouple the impacts of distinct events implicated in GCN2 activation, and to monitor the consequences of different translational stresses. Additionally, ribosome collisions induced by HF in this system are rapidly reversed by resupply of proline, which permits the study of signaling events linked to translational restart during recovery from amino acid stress. RESULTS: We found that the initiation of GCN2 signaling by different cellular stresses shared the following central requirements: (i) ribosome collisions, (ii) GCN1 recruitment to collided ribosomes, (iii) GCN1-dependent recruitment of GCN2 to collided ribosomes, and (iv) specific GCN2 interactions with collided ribosomes. In contrast to prior models that proposed a direct role for uncharged tRNA in GCN2 activation, we found that an increase in uncharged tRNA was neither required nor sufficient to trigger the initiation of GCN2 signaling in response to amino acid stress. Instead, amino acid stress–induced activation of GCN2 involved a specific signaling refinement: Uncharged tRNA cognate to the A-site codon of the lead, stalled ribosome enhances GCN2 activation. CONCLUSION: These findings define the core mechanistic requirements for GCN2 activation and highlight the importance of ribosome collisions as a shared feature of a broad range of cellular stress responses. Moreover, they provide a unifying model for GCN2 activation that helps to explain how multiple inputs, previously individually implicated in GCN2 activation, contribute to GCN2 signaling. GCN2 activation on collided ribosomes.: GCN2 is activated by multiple translational stresses, affecting a range of physiological functions. GCN2 activation on collided ribosomes requires GCN1 recruitment of GCN2 to collisions and interactions between GCN2 and the lead, stalled ribosome. Specific to amino acid stress, GCN2 signaling is enhanced by decoding of uncharged tRNA that is matched to the A-site of the lead, stalled ribosome. [ABSTRACT FROM AUTHOR]