Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells

Hinnebusch AG (2014) Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2a Phosphorylation in Starved Cells. PLoS Genet 10(5): e1004326. doi:10.1371/journal.pgen.1004326 Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2a Phosphorylation in Starved Cells Sebastien Lageix 0 Stefan Rothenburg 0 Thomas E. Dever 0 Alan G. Hinnebusch 0 Mark Ashe, University of Manchester, United Kingdom 0 1 Laboratory of Gene Regulation and Development, Eunice K. Shriver National Institute of Child Health and Human Development, National Institutes of Health , Bethesda , Maryland, United States of America, 2 Kansas State University, Division of Biology , Manhattan, Kansas , United States of America 1 www.plosgenetics.org The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2a, from yeast to mammals. The Gcn2 kinase domain (KD) is inherently inactive and requires allosteric stimulation by adjoining regulatory domains. Gcn2 contains a pseudokinase domain (YKD) required for high-level eIF2a phosphorylation in amino acid starved yeast cells; however, the role of the YKD in KD activation was unknown. We isolated substitutions of evolutionarily conserved YKD amino acids that impair Gcn2 activation without reducing binding of the activating ligand, uncharged tRNA, to the histidyl-tRNA synthetase-related domain of Gcn2. Several such Gcn2 substitutions cluster in predicted helices E and I (aE and aI) of the YKD. We also identified Gcd2 substitutions, evoking constitutive activation of Gcn2, mapping in aI of the YKD. Interestingly, aI Gcd2 substitutions enhance YKD-KD interactions in vitro, whereas Gcn2 substitutions in aE and aI suppress both this effect and the constitutive activation of Gcn2 conferred by YKD Gcd2 substitutions. These findings indicate that the YKD interacts directly with the KD for activation of kinase function and identify likely sites of direct YKD-KD contact. We propose that tRNA binding to the HisRS domain evokes a conformational change that increases access of the YKD to sites of allosteric activation in the adjoining KD. - Funding: This work was supported by the Intramural Research Program of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Eukaryotic cells harbor stress-activated protein kinases that down-regulate protein synthesis and simultaneously up-regulate transcriptional activators at the translational level. This dual response allows cells to reduce bulk protein synthesis while reprogramming transcription to favor expression of gene products with functions in stress management. The key target of these kinases is Ser-51 of the a-subunit of translation initiation factor 2 (eIF2a). The eIF2 bound to GTP transfers methionyl-initiator tRNA to the 40S ribosomal subunit to produce the 43S preinitiation complex at the beginning of the translation initiation pathway. On subsequent recognition of the AUG codon in mRNA by initiator tRNA, the GTP is hydrolyzed and eIF2-GDP is released from the 40S subunit for recycling to eIF2-GTP by the guanine nucleotide exchange factor eIF2B. Ser-51 phosphorylation converts eIF2 into an inhibitor of eIF2B, reducing the concentration of eIF2-GTP and delaying new rounds of translation initiation. The reduced eIF2-GTP level stimulates translation of GCN4 mRNA in yeast and ATF4 mRNA in mammals, both encoding transcriptional activators of stress genes, by allowing 43S complexes to circumvent small open reading frames present in their mRNA leaders that would normally block initiation at the protein coding sequences for Gcn4/Atf4 [1,2] (reviewed in [3]). The four mammalian eIF2a kinases, PKR, HRI, PERK, and Gcn2, have conserved kinase domains (KDs) but unique regulatory regions that mediate activation by distinct stress signals. PKR is activated by dsRNA generated during virus infection, and represents a key component of the antiviral defense mechanism, whereas Gcn2 is activated by uncharged tRNA that accumulates in amino acid-starved cells and most likely other stress conditions. The ensuing induction of Gcn4 in yeast evokes transcriptional activation of nearly all amino acid biosynthetic enzymes subject to the general amino acid control with attendant up-regulation of amino acid biosynthesis (reviewed in [3]). Translational control by mammalian Gcn2 is important for lipid homeostasis under starvation conditions [4], in behavioral aversion to amino aciddeficient diets [5], and in learning and memory [6]. It has also been implicated in tumor cell survival, both innate and T-cell mediated immune responses, and DNA repair upon UV irradiation (reviewed in [7]). Because eIF2a kinases act by inhibiting translation, their functions must be tightly regulated to allow high-level kinase activity only under appropriate stress conditions. We showed previously that the Gcn2 KD is intrinsically inert and depends on stimulatory interactions with adjacent domains in the protein to achieve an active conformation [8]. This latency of Gcn2 depends on a rigid hinge connecting the N- and C-lobes, which promotes a partially closed active site cleft and occluded ATP-binding pocket, The survival of all living organisms depends on their capacity to adapt their gene expression program to variations in the environment. When subjected to various stresses, eukaryotic cells down-regulate general protein synthesis by phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2a). The yeast Saccharomyces cerevisiae has a single eIF2a kinase, Gcn2, activated by uncharged tRNAs accumulating in amino acid starved cells, which bind to a regulatory domain homologous to histidyl-tRNA synthetase. Gcn2 also contains a degenerate, pseudokinase domain (YKD) of largely unknown function, juxtaposed to the authentic, functional kinase domain (KD). Our study demonstrates that direct interaction between the YKD and KD is essential for activation of Gcn2, and identifies likely KD-contact sites in the YKD that can be altered to either impair or constitutively activate kinase function. Our results provide the first functional insights into the regulatory role of the enigmatic YKD of Gcn2. and a non-productive orientation of helix aC in the N-lobe that impedes proper disposition of a critical Lys reside that positions the ATP phosphates for catalysis [9,10]. Binding of uncharged tRNA to a region C-terminal to the KD, related in sequence to the enzyme histidyl-tRNA synthetase (HisRS), which aminoacylates tRNAHis, is required to activate Gcn2 in amino acid-starved cells [11,12,13,14]. An N-terminal segment in the HisRS domain that interacts with a portion of the KD containing the hinge is required for kinase activation [15], suggesting that tRNA bind (...truncated)