Ribosome-binding Domain of Eukaryotic Initiation Factor-2 Kinase GCN2 Facilitates Translation Control

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 ␣ 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 doublestranded 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.

Crystal Structures of GCN2 Protein Kinase C-terminal Domains Suggest Regulatory Differences in Yeast and Mammals

“…Other proposed mechanistic features mediated by the CTD are dimerization and ribosome binding. The CTD of yeast GCN2 was shown to be sufficient to mediate binding of this eIF2 kinase to ribosomes as judged by co-migration with ribosomes separated by sucrose gradient centrifugation (19,21,24). In yeast, GCN2 association with the translational machinery has been proposed to be important for facilitating binding to accumulating uncharged tRNAs in the context of the A site of ribosomes (25).…”

“…In yeast, GCN2 association with the translational machinery has been proposed to be important for facilitating binding to accumulating uncharged tRNAs in the context of the A site of ribosomes (25). Furthermore, the CTD of GCN2 can bind double-stranded (ds) RNA through a cluster of lysine residues, which had led to the proposal that GCN2 associates with ribosomes through direct interactions with rRNA (21). The sequences of the CTD of GCN2 in vertebrates share little sequence similarity with their yeast counterpart; therefore, it is not currently known whether the functional features attributed to the yeast GCN2 CTD are functionally germane to mechanisms regulating GCN2 phosphorylation of eIF2 in mammals.…”

YIH1 Is an Actin-binding Protein That Inhibits Protein Kinase GCN2 and Impairs General Amino Acid Control When Overexpressed

“…GCN1 and GCN2 can interact independently with ribosomes, and these interactions are thought to be crucial for activation of GCN2 in starved cells (8,15,17). Genetic evidence further suggests that GCN1 binds near the decoding (A) site of the ribosome.…”

“…The uncharged tRNA binds to a regulatory domain in GCN2 that resembles histidyl-tRNA synthetase, and this interaction is believed to induce a conformational change that overcomes an intrinsic defect in the adjacent kinase domain (7)(8)(9)(10). The products of GCN1 and GCN20 are also necessary for the activation of GCN2 by uncharged tRNA in starved cells (11,12).…”

Serine 577 Is Phosphorylated and Negatively Affects the tRNA Binding and eIF2α Kinase Activities of GCN2

“…The extreme C-terminal segment of GCN2 (Cterm) also is needed for strong tRNA binding in vitro, suggesting that GCN2 contains a bipartite tRNA-binding surface (11). The Cterm is additionally required for dimerization (15,16) and ribosome binding by GCN2 in cell extracts (17,18) (Fig. 1).…”