A Network of Hydrophobic Residues Impeding Helix αC Rotation Maintains Latency of Kinase Gcn2, Which Phosphorylates the α Subunit of Translation Initiation Factor 2

Abstract

Kinase Gcn2 is activated by amino acid starvation and downregulates translation initiation by phosphorylating the ␣ subunit of translation initiation factor 2 (eIF2␣). The Gcn2 kinase domain (KD) is inert and must be activated by tRNA binding to the adjacent regulatory domain. Previous work indicated that Saccharomyces cerevisiae Gcn2 latency results from inflexibility of the hinge connecting the N and C lobes and a partially obstructed ATP-binding site in the KD. Here, we provide strong evidence that a network of hydrophobic interactions centered on Leu-856 also promotes latency by constraining helix ␣C rotation in the KD in a manner relieved during amino acid starvation by tRNA binding and autophosphorylation of Thr-882 in the activation loop. Thus, we show that mutationally disrupting the hydrophobic network in various ways constitutively activates eIF2␣ phosphorylation in vivo and bypasses the requirement for a key tRNA binding motif (m2) and Thr-882 in Gcn2. In particular, replacing Leu-856 with any nonhydrophobic residue activates Gcn2, while substitutions with various hydrophobic residues maintain kinase latency. We further provide strong evidence that parallel, back-to-back dimerization of the KD is a step on the Gcn2 activation pathway promoted by tRNA binding and autophosphorylation. Remarkably, mutations that disrupt the L856 hydrophobic network or enhance hinge flexibility eliminate the need for the conserved salt bridge at the parallel dimer interface, implying that KD dimerization facilitates the reorientation of ␣C and remodeling of the active site for enhanced ATP binding and catalysis. We propose that hinge remodeling, parallel dimerization, and reorientation of ␣C are mutually reinforcing conformational transitions stimulated by tRNA binding and secured by the ensuing autophosphorylation of T882 for stable kinase activation.

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…Gcn2 is one of a family of protein kinases that phosphorylate eIF2a on Ser51 to regulate global protein synthesis across eukaryotes (Wek et al 2006;Dever et al 2007). In its basal state, the Gcn2 kinase is inactive, and in response to an activation signal, conformational changes and altered interactions within the multidomain protein enable its eIF2a kinase activity (Qiu et al 2001(Qiu et al , 2002Padyana et al 2005;Garriz et al 2009;Lageix et al 2014).…”

“…Gcn2 is a dimer with multiple intermolecular interactions between monomers as well as intramolecular interactions between adjacent domains and longer range interactions between the KD and the RBD within Gcn2 monomers (Ramirez et al 1992;Zhu et al 1996;Qiu et al 1998Qiu et al , 2001Padyana et al 2005;Garriz et al 2009;Lageix et al 2014). Binding of uncharged tRNA to the HisRS domain (Zhu et al 1996;Dong et al 2000) stimulates autophosphorylation of the activation loop in the KD on Thr882 and Thr887 (Romano et al 1998) enabling phosphorylation of its only known substrate eIF2a (Dey et al 2005(Dey et al , 2007Padyana et al 2005).…”

Perturbations in actin dynamics reconfigure protein complexes that modulate GCN2 activity and promote an eIF2 response

“…Under nutrient-replete conditions it is kept in a latent state by several autoinhibitory interactions (Garriz et al, 2009;Lageix et al, 2015). Uncharged tRNA that accumulates under amino acid depletion or other stress conditions binds to a region in GCN2 with homology to histidyl tRNA synthetases (HisRS).…”

Structural analysis of the EGFR TK domain and potential implications for EGFR targeted therapy

“…There is a broad consensus on the mechanism of activating mutations that the stabilization of the hydrophobic regulatory spine promotes shift of the kinase towards the constitutively active kinase form, and thus have a dramatic effect on the regulation of the enzyme (16). Because different mutations lead to different sensitivities to TKI, we analyzed and compared all the available structures of EGFR, aiming at demonstrating how structural insights help our understanding of active and resistance mechanisms.…”