Abstract

A family of protein kinases regulate translation initiation in response to cellular stresses by phosphorylation of eukaryotic initiation factor-2 (eIF-2). One family member from yeast, GCN2, contains a region homologous to histidyl-tRNA synthetases juxtaposed to the kinase catalytic domain. It is thought that uncharged tRNA accumulating during amino acid starvation binds to the synthetase-related sequences and stimulates phosphorylation of the alpha subunit of eIF-2. In this report, we define another domain in GCN2 that functions to target the kinase to ribosomes. A truncated version of GCN2 containing only amino acid residues 1467 to 1590 can independently associate with the translational machinery. Interestingly, this region of GCN2 shares sequence similarities with the core of the double-stranded RNA-binding domain (DRBD). Substitutions of the lysine residues conserved among DRBD sequences block association of GCN2 with ribosomes and impaired the ability of the kinase to stimulate translational control in response to amino acid limitation. Additionally, as found for other DRBD sequences, recombinant protein containing GCN2 residues 1467-1590 can bind double-stranded RNA in vitro, suggesting that interaction with rRNA mediates ribosome targeting. These results indicate that appropriate ribosome localization of the kinase is an obligate step in the mechanism leading to translational control by GCN2.

Highlights

  • Targeting of proteins to different compartments in the cell is an important mechanism regulating protein function

  • Substitutions of lysine residues conserved among double-stranded RNA-binding domain (DRBD) block association of GCN2 with ribosomes and impaired the ability of the kinase to stimulate the general control pathway in response to amino acid limitation

  • These results suggest that appropriate localization of GCN2 to the translational machinery is an obligate step in the mechanism leading to kinase phosphorylation of eukaryotic translation initiation factor-2 (eIF-2)

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Summary

Introduction

Targeting of proteins to different compartments in the cell is an important mechanism regulating protein function. It is proposed that different uncharged tRNAs, which accumulate during amino acid starvation conditions, can interact with the synthetase-related domain of GCN2, resulting in activation of the kinase and phosphorylation of eIF-2 [2, 4, 12, 15, 16] Another domain that is important for regulation of GCN2 involves targeting of the kinase to ribosomes. As previously observed for the DRBDs, the lysine residues are essential for binding to dsRNA in vitro, suggesting that interaction with rRNA mediates ribosome targeting This cellular localization may provide GCN2 access to its eIF-2 substrate or to regulatory ligands. These findings indicate that related RNA-binding sequences facilitates the in vivo activities of both GCN2 and PKR in response to cellular stresses

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