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J. Biol. Chem., Vol. 279, Issue 22, 22820-22832, May 28, 2004

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Dimerization Is Required for Activation of eIF2 Kinase Gcn2 in Response to Diverse Environmental Stress Conditions^*

Jana Narasimhan, Kirk A. Staschke, and Ronald C. Wek

From the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202

In the yeast Saccharomyces cerevisiae, starvation for amino acids induces phosphorylation of the subunit of eukaryotic initiation factor 2 by Gcn2 protein kinase, leading to elevated translation of GCN4. Gcn4p is a transcriptional activator of hundreds of genes involved in remedying nutrient deprivation. In addition to a conserved kinase domain, Gcn2p has a regulatory region homologous to histidyl tRNA synthetase enzymes that binds uncharged tRNA that accumulates during amino acid starvation. Flanking the carboxyl terminus of the histidyl-tRNA synthetase-related domain is a region spanning 162 residues that participates in the activation of the protein kinase. Gel filtration and chemical cross-linking analysis of the recombinant carboxyl-terminal Gcn2 protein revealed that this region is a stable homodimer that is highly resistant to high concentrations of salt. Residue alterations in three hydrophobic segments and one segment with a proposed amphipathic -helix in this Gcn2p carboxyl terminus blocked oligomerization, supporting the role of hydrophobic interactions in the dimerization interface of Gcn2p. Introduction of residue substitutions that impaired dimerization into the full-length protein prevented the ability of Gcn2p to phosphorylate its substrate eukaryotic initiation factor-2 and induce GCN4 translational expression in yeast cells subjected to a variety of stresses including amino acid limitation or exposure to rapamycin or high levels of NaCl. This latter stress can be overcome by addition of increasing amounts of K⁺ ions, indicating that the Na⁺/K⁺ ion balance is central to this stress induction. We conclude that dimerization involving hydrophobic segments in the carboxyl-terminal region is required for activation of Gcn2p in response to a multitude of stresses.

Received for publication, February 27, 2004

^* This work was supported in part by Grants R01GM49164 and R01GM643540 (to R. C. W.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 317-274-0549; Fax: 317-274-4686; E-mail: rwek{at}iupui.edu.

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