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GCN2 Whets the Appetite for Amino Acids
Molecular Cell, Vol. 18, 141–148, April 15, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.molcel.2005.03.023
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GCN2 Whets the Appetite for Amino Acids In response to amino acid starvation, the kinase GCN2 in yeast activates amino acid biosynthesis. Two recent studies (Maurin et al., 2005; Hao et al., 2005) reveal that GCN2 in the brain of mice restricts intake of diets lacking essential amino acids. Omnivorous animals select foods to achieve a balanced diet that includes adequate levels of the essential amino acids. Although it is known that rats will reject diets lacking an essential amino acid, the molecular mechanisms governing this feeding behavior are not understood. In contrast to animals, the yeast Saccharomyces cerevisiae can synthesize all 20 amino acids and grow in medium lacking these nutrients. One can interfere with the ability of yeast to synthesize an amino acid by mutating or inhibiting a biosynthetic enzyme, or by culturing yeast in an unbalanced medium where an amino acid present in excess feedback inhibits an enzyme involved in biosynthesis of a second amino acid lacking in the medium. Each of these conditions triggers the response known as general amino acid control (GAAC, Figure 1), a crosspathway regulation of amino acid biosynthesis. When starved for any of several amino acids, yeast cells induce the transcription of over 70 genes encoding enzymes that function in the biosynthesis of all 20 amino acids (Hinnebusch and Natarajan, 2002). The signal for amino acid starvation in yeast cells is uncharged tRNA, as mutation of an aminoacyl-tRNA synthetase triggers GAAC even in medium replete with amino acids. Genetic studies in yeast identified the protein kinase GCN2 as the primary sensor of amino acid starvation, and the domain structure of GCN2 (Figure 1) supports this assignment. In addition to a typical eukaryotic protein kinase domain, GCN2 contains a domain related to histidyl-tRNA synthetase (HisRS), which mediates GCN2 activation in response to starvation for different amino acids (Wek et al., 1995) and which binds different uncharged tRNAs in vitro with higher affinity than the corresponding charged tRNA (Dong et al., 2000). Binding of uncharged tRNA to the HisRS domain is thought to activate the adjacent protein kinase domain, whose only known substrate is the GTP binding translation initiation factor eIF2. Phosphorylation of the α subunit of eIF2 on serine 51 in yeast requires GCN2 and is enhanced in cells grown under amino acid starvation conditions (Dever et al., 1992). The eIF2 forms a ternary complex (TC) with GTP and the initiator methionyl-tRNA (Met-tRNA) and delivers Met-tRNA to the small ribosomal subunit in the first step of translation initiation. The resulting ribosomal complex binds to mRNA, scans to an AUG initiation codon, and releases eIF2-GDP after GTP hydrolysis. The guanine nucleotide exchange factor eIF2B recycles eIF2•GDP to functional eIF2•GTP, and this reaction is blocked by eIF2α phosphorylation.
Thus, phosphorylation of eIF2α by GCN2 in starved cells inhibits protein synthesis and limits consumption of amino acids. In addition to reducing protein synthesis, eIF2α phosphorylation induces translation of GCN4, the primary regulator of GAAC. GCN4 is the transcription factor that both binds to promoters and activates transcription of the myriad biosynthetic genes subject to GAAC (Hinnebusch and Natarajan, 2002). Both GCN2 and eIF2α phosphorylation are required for increased synthesis of GCN4 under starvation conditions, and this translational control is mediated by regulated reinitiation at
Figure 1. Central Role for GCN2 in Maintaining Amino Acid Homeostasis in Yeast and Animals Amino acid starvation causes the accumulation of uncharged tRNAs that bind to the histidyl-tRNA synthetase (HisRS)-related domain of GCN2 and activate the adjacent protein kinase domain (KD). As indicated, the w180 kDa GCN2 also contains a pseudoprotein kinase domain (ΨKD) of unknown function and an N-terminal binding site for the GCN1/GCN20 complex that is essential for recognition of the starvation signal (Hinnebusch and Natarajan, 2002). GCN2 phosphorylates the translation factor eIF2α on serine 51, converting eIF2 into an inhibitor of its guanine nucleotide exchange factor eIF2B and leading to inhibition of protein synthesis. Paradoxically, phosphorylation of eIF2α and inhibition of eIF2B leads to the translational induction of GCN4 synthesis in yeast, and GCN4 activates the transcription of genes encoding enzymes that function in amino acid biosynthesis. In mice, activation of GCN2 in the anterior piriform cortex of the brain mediates an aversive response toward intake of diets lacking an essential amino acid (AA). As indicated (?), the components of the pathway linking eIF2α phosphorylation and eating behavior in animals are not known, but the transcription factor ATF4 is a possible candidate.
Molecular Cell 142
short, upstream open reading frames (uORFs) in the GCN4 mRNA leader. In contrast to yeast, animals can synthesize only a subset of amino acids and must obtain the essential or indispensable amino acids (IAAs) from diet. It was known that rats and other omnivores reject diets lacking a single IAA, and ablation studies revealed that the anterior piriform cortex (APC) in the brain is critical for this behavioral response. Direct injection of the limiting amino acid into the APC blocks the rejection of deficient diets. Although several pathways important for neuronal signaling are apparently activated in response to diets lacking an IAA, the molecules responsible for sensing amino acid imbalance and directing eating behavior have not been identified until now. Recently, Gietzen and colleagues (Gietzen et al., 2004) reported elevated eIF2α phosphorylation on serine 51 in the APC of rats fed a threonine-deficient diet. GCN2 is conserved in all eukaryotes and is activated by amino acid limitation in cultured mouse cells (Harding et al., 2000). This fact, the conspicuous presence of GCN2 in mammalian brain, and the demonstration that GCN2 mediates increased eIF2α phosphorylation in the liver of mice fed diets lacking leucine, but not the nonessential amino acid glycine (Anthony et al., 2004), all led to the hypothesis that GCN2 senses amino acid imbalance and initiates a signal transduction pathway leading to rejection of IAA-deficient diets. In two recent reports, Hao et al. (2005) and Maurin et al. (2005) test this hypothesis by examining mice lacking GCN2. Hao and colleagues found that injecting the APC with the amino alcohol threoninol, which blocks threonyl-tRNA charging, caused wild-type (GCN2+/+) mice to reject diets with basal levels of threonine, but not threonine-replete diets (Hao et al., 2005). Thus, uncharged tRNA, which is the activating signal for GCN2 in yeast, is likely the signal for amino acid imbalance that triggers the eating response in mice. Both groups report increased eIF2α serine 51 phosphorylation in the APC of GCN2+/+, but not GCN2−/−, mice 20 min after ingestion of a meal lacking an IAA. Moreover, Maurin and colleagues observed enhanced GCN2 autophosphorylation, consistent with kinase activation, and eIF2α phosphorylation in the liver of GCN2+/+, but not GCN2−/−, mice fed a diet lacking the essential amino acid tryptophan (Maurin et al., 2005). The two groups extended the analysis by examining feeding behavior. Whereas GCN2+/+ mice readily reject a diet lacking leucine or threonine, the GCN2−/− mice have a significantly blunted aversive response and, in some cases, consume more of the amino acid-deficient diet than the balanced diet. Importantly, Maurin and colleagues report that a brain-specific knockout of GCN2 impairs the aversive response to the amino aciddeficient diet, but not the phosphorylation of eIF2α in liver (Maurin et al., 2005). The behavior of the mice lacking GCN2 specifically in the brain or injected with amino alcohols in the APC strongly indicates that recognition of uncharged tRNA and eIF2α phosphorylation by GCN2 in APC neurons is critical for rejection of diets lacking an IAA. Thus, GCN2 has a conserved function to maintain amino acid homeostasis by modu-
lating amino acid biosynthesis in yeast and by governing feeding behavior in mammals. The mouse liver also senses IAA limitation in the diet, and GCN2 additionally functions in this organ to suppress protein synthesis as a way of sparing muscle mass in starved animals (Anthony et al., 2004). The exciting findings in these papers raise several intriguing questions. Although eIF2α is the only identified substrate of GCN2, it is formally possible that another substrate is important for this behavioral response. Thus, would overexpression of a nonphosphorylatable version of eIF2α (serine 51 to alanine) in the brain of mice phenocopy the GCN2 knockout and elicit enhanced consumption of unbalanced diets? Would activation of another eIF2α kinase, such as the endoplasmic reticulum stress-responsive PERK (Harding et al., 2000), prevent animals from selectively rejecting IAA-deficient diets? Are the mammalian counterparts of GCN1 and GCN20, positive regulators of GCN2 in yeast (Hinnebusch and Natarajan, 2002), required for the behavioral response? Finally, how does phosphorylation of eIF2α elicit rejection of unbalanced diets? Analogous to translational control of GCN4 in yeast, expression of the transcription factor ATF4 is coupled to eIF2α phosphorylation through regulated reinitiation at uORFs in the ATF4 mRNA leader (Harding et al., 2000; Vattem and Wek, 2004). It will be interesting to learn whether ATF4 or another translationally regulated mRNA links eIF2α phosphorylation with downstream effectors governing feeding behavior in animals. Thomas E. Dever and Alan G. Hinnebusch Laboratory of Gene Regulation and Development National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20892
Selected Reading Anthony, T.G., McDaniel, B.J., Byerley, R.L., McGrath, B.C., Cavener, D.R., McNurlan, M.A., and Wek, R.C. (2004). J. Biol. Chem. 279, 36553–36561. Dever, T.E., Feng, L., Wek, R.C., Cigan, A.M., Donahue, T.D., and Hinnebusch, A.G. (1992). Cell 68, 585–596. Dong, J., Qiu, H., Garcia-Barrio, M., Anderson, J., and Hinnebusch, A.G. (2000). Mol. Cell 6, 269–279. Gietzen, D.W., Ross, C.M., Hao, S., and Sharp, J.W. (2004). J. Nutr. 134, 717–723. Hao, S., Sharp, J.W., Ross-Inta, C.M., McDaniel, B.J., Anthony, T.G., Wek, R.C., Cavener, D.R., McGrath, B.C., Rudell, J.B., Koehnle, T.J., and Gietzen, D.W. (2005). Science 307, 1776–1778. Harding, H.P., Novoa, I., Zhang, Y., Zeng, H., Wek, R., Schapira, M., and Ron, D. (2000). Mol. Cell 6, 1099–1108. Hinnebusch, A.G., and Natarajan, K. (2002). Eukaryot. Cell 1, 22–32. Maurin, A.-C., Jousse, C., Averous, J., Parry, L., Bruhat, A., Cherasse, Y., Zeng, H., Zhang, Y., Harding, H.P., Ron, D., and Fafournoux, P. (2005). Cell Metab. 1, 273–277. Vattem, K.M., and Wek, R.C. (2004). Proc. Natl. Acad. Sci. USA 101, 11269–11274. Wek, S.A., Zhu, S., and Wek, R.C. (1995). Mol. Cell. Biol. 15, 4497– 4506.
Previews
GCN2 Whets the Appetite for Amino Acids In response to amino acid starvation, the kinase GCN2 in yeast activates amino acid biosynthesis. Two recent studies (Maurin et al., 2005; Hao et al., 2005) reveal that GCN2 in the brain of mice restricts intake of diets lacking essential amino acids. Omnivorous animals select foods to achieve a balanced diet that includes adequate levels of the essential amino acids. Although it is known that rats will reject diets lacking an essential amino acid, the molecular mechanisms governing this feeding behavior are not understood. In contrast to animals, the yeast Saccharomyces cerevisiae can synthesize all 20 amino acids and grow in medium lacking these nutrients. One can interfere with the ability of yeast to synthesize an amino acid by mutating or inhibiting a biosynthetic enzyme, or by culturing yeast in an unbalanced medium where an amino acid present in excess feedback inhibits an enzyme involved in biosynthesis of a second amino acid lacking in the medium. Each of these conditions triggers the response known as general amino acid control (GAAC, Figure 1), a crosspathway regulation of amino acid biosynthesis. When starved for any of several amino acids, yeast cells induce the transcription of over 70 genes encoding enzymes that function in the biosynthesis of all 20 amino acids (Hinnebusch and Natarajan, 2002). The signal for amino acid starvation in yeast cells is uncharged tRNA, as mutation of an aminoacyl-tRNA synthetase triggers GAAC even in medium replete with amino acids. Genetic studies in yeast identified the protein kinase GCN2 as the primary sensor of amino acid starvation, and the domain structure of GCN2 (Figure 1) supports this assignment. In addition to a typical eukaryotic protein kinase domain, GCN2 contains a domain related to histidyl-tRNA synthetase (HisRS), which mediates GCN2 activation in response to starvation for different amino acids (Wek et al., 1995) and which binds different uncharged tRNAs in vitro with higher affinity than the corresponding charged tRNA (Dong et al., 2000). Binding of uncharged tRNA to the HisRS domain is thought to activate the adjacent protein kinase domain, whose only known substrate is the GTP binding translation initiation factor eIF2. Phosphorylation of the α subunit of eIF2 on serine 51 in yeast requires GCN2 and is enhanced in cells grown under amino acid starvation conditions (Dever et al., 1992). The eIF2 forms a ternary complex (TC) with GTP and the initiator methionyl-tRNA (Met-tRNA) and delivers Met-tRNA to the small ribosomal subunit in the first step of translation initiation. The resulting ribosomal complex binds to mRNA, scans to an AUG initiation codon, and releases eIF2-GDP after GTP hydrolysis. The guanine nucleotide exchange factor eIF2B recycles eIF2•GDP to functional eIF2•GTP, and this reaction is blocked by eIF2α phosphorylation.
Thus, phosphorylation of eIF2α by GCN2 in starved cells inhibits protein synthesis and limits consumption of amino acids. In addition to reducing protein synthesis, eIF2α phosphorylation induces translation of GCN4, the primary regulator of GAAC. GCN4 is the transcription factor that both binds to promoters and activates transcription of the myriad biosynthetic genes subject to GAAC (Hinnebusch and Natarajan, 2002). Both GCN2 and eIF2α phosphorylation are required for increased synthesis of GCN4 under starvation conditions, and this translational control is mediated by regulated reinitiation at
Figure 1. Central Role for GCN2 in Maintaining Amino Acid Homeostasis in Yeast and Animals Amino acid starvation causes the accumulation of uncharged tRNAs that bind to the histidyl-tRNA synthetase (HisRS)-related domain of GCN2 and activate the adjacent protein kinase domain (KD). As indicated, the w180 kDa GCN2 also contains a pseudoprotein kinase domain (ΨKD) of unknown function and an N-terminal binding site for the GCN1/GCN20 complex that is essential for recognition of the starvation signal (Hinnebusch and Natarajan, 2002). GCN2 phosphorylates the translation factor eIF2α on serine 51, converting eIF2 into an inhibitor of its guanine nucleotide exchange factor eIF2B and leading to inhibition of protein synthesis. Paradoxically, phosphorylation of eIF2α and inhibition of eIF2B leads to the translational induction of GCN4 synthesis in yeast, and GCN4 activates the transcription of genes encoding enzymes that function in amino acid biosynthesis. In mice, activation of GCN2 in the anterior piriform cortex of the brain mediates an aversive response toward intake of diets lacking an essential amino acid (AA). As indicated (?), the components of the pathway linking eIF2α phosphorylation and eating behavior in animals are not known, but the transcription factor ATF4 is a possible candidate.
Molecular Cell 142
short, upstream open reading frames (uORFs) in the GCN4 mRNA leader. In contrast to yeast, animals can synthesize only a subset of amino acids and must obtain the essential or indispensable amino acids (IAAs) from diet. It was known that rats and other omnivores reject diets lacking a single IAA, and ablation studies revealed that the anterior piriform cortex (APC) in the brain is critical for this behavioral response. Direct injection of the limiting amino acid into the APC blocks the rejection of deficient diets. Although several pathways important for neuronal signaling are apparently activated in response to diets lacking an IAA, the molecules responsible for sensing amino acid imbalance and directing eating behavior have not been identified until now. Recently, Gietzen and colleagues (Gietzen et al., 2004) reported elevated eIF2α phosphorylation on serine 51 in the APC of rats fed a threonine-deficient diet. GCN2 is conserved in all eukaryotes and is activated by amino acid limitation in cultured mouse cells (Harding et al., 2000). This fact, the conspicuous presence of GCN2 in mammalian brain, and the demonstration that GCN2 mediates increased eIF2α phosphorylation in the liver of mice fed diets lacking leucine, but not the nonessential amino acid glycine (Anthony et al., 2004), all led to the hypothesis that GCN2 senses amino acid imbalance and initiates a signal transduction pathway leading to rejection of IAA-deficient diets. In two recent reports, Hao et al. (2005) and Maurin et al. (2005) test this hypothesis by examining mice lacking GCN2. Hao and colleagues found that injecting the APC with the amino alcohol threoninol, which blocks threonyl-tRNA charging, caused wild-type (GCN2+/+) mice to reject diets with basal levels of threonine, but not threonine-replete diets (Hao et al., 2005). Thus, uncharged tRNA, which is the activating signal for GCN2 in yeast, is likely the signal for amino acid imbalance that triggers the eating response in mice. Both groups report increased eIF2α serine 51 phosphorylation in the APC of GCN2+/+, but not GCN2−/−, mice 20 min after ingestion of a meal lacking an IAA. Moreover, Maurin and colleagues observed enhanced GCN2 autophosphorylation, consistent with kinase activation, and eIF2α phosphorylation in the liver of GCN2+/+, but not GCN2−/−, mice fed a diet lacking the essential amino acid tryptophan (Maurin et al., 2005). The two groups extended the analysis by examining feeding behavior. Whereas GCN2+/+ mice readily reject a diet lacking leucine or threonine, the GCN2−/− mice have a significantly blunted aversive response and, in some cases, consume more of the amino acid-deficient diet than the balanced diet. Importantly, Maurin and colleagues report that a brain-specific knockout of GCN2 impairs the aversive response to the amino aciddeficient diet, but not the phosphorylation of eIF2α in liver (Maurin et al., 2005). The behavior of the mice lacking GCN2 specifically in the brain or injected with amino alcohols in the APC strongly indicates that recognition of uncharged tRNA and eIF2α phosphorylation by GCN2 in APC neurons is critical for rejection of diets lacking an IAA. Thus, GCN2 has a conserved function to maintain amino acid homeostasis by modu-
lating amino acid biosynthesis in yeast and by governing feeding behavior in mammals. The mouse liver also senses IAA limitation in the diet, and GCN2 additionally functions in this organ to suppress protein synthesis as a way of sparing muscle mass in starved animals (Anthony et al., 2004). The exciting findings in these papers raise several intriguing questions. Although eIF2α is the only identified substrate of GCN2, it is formally possible that another substrate is important for this behavioral response. Thus, would overexpression of a nonphosphorylatable version of eIF2α (serine 51 to alanine) in the brain of mice phenocopy the GCN2 knockout and elicit enhanced consumption of unbalanced diets? Would activation of another eIF2α kinase, such as the endoplasmic reticulum stress-responsive PERK (Harding et al., 2000), prevent animals from selectively rejecting IAA-deficient diets? Are the mammalian counterparts of GCN1 and GCN20, positive regulators of GCN2 in yeast (Hinnebusch and Natarajan, 2002), required for the behavioral response? Finally, how does phosphorylation of eIF2α elicit rejection of unbalanced diets? Analogous to translational control of GCN4 in yeast, expression of the transcription factor ATF4 is coupled to eIF2α phosphorylation through regulated reinitiation at uORFs in the ATF4 mRNA leader (Harding et al., 2000; Vattem and Wek, 2004). It will be interesting to learn whether ATF4 or another translationally regulated mRNA links eIF2α phosphorylation with downstream effectors governing feeding behavior in animals. Thomas E. Dever and Alan G. Hinnebusch Laboratory of Gene Regulation and Development National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20892
Selected Reading Anthony, T.G., McDaniel, B.J., Byerley, R.L., McGrath, B.C., Cavener, D.R., McNurlan, M.A., and Wek, R.C. (2004). J. Biol. Chem. 279, 36553–36561. Dever, T.E., Feng, L., Wek, R.C., Cigan, A.M., Donahue, T.D., and Hinnebusch, A.G. (1992). Cell 68, 585–596. Dong, J., Qiu, H., Garcia-Barrio, M., Anderson, J., and Hinnebusch, A.G. (2000). Mol. Cell 6, 269–279. Gietzen, D.W., Ross, C.M., Hao, S., and Sharp, J.W. (2004). J. Nutr. 134, 717–723. Hao, S., Sharp, J.W., Ross-Inta, C.M., McDaniel, B.J., Anthony, T.G., Wek, R.C., Cavener, D.R., McGrath, B.C., Rudell, J.B., Koehnle, T.J., and Gietzen, D.W. (2005). Science 307, 1776–1778. Harding, H.P., Novoa, I., Zhang, Y., Zeng, H., Wek, R., Schapira, M., and Ron, D. (2000). Mol. Cell 6, 1099–1108. Hinnebusch, A.G., and Natarajan, K. (2002). Eukaryot. Cell 1, 22–32. Maurin, A.-C., Jousse, C., Averous, J., Parry, L., Bruhat, A., Cherasse, Y., Zeng, H., Zhang, Y., Harding, H.P., Ron, D., and Fafournoux, P. (2005). Cell Metab. 1, 273–277. Vattem, K.M., and Wek, R.C. (2004). Proc. Natl. Acad. Sci. USA 101, 11269–11274. Wek, S.A., Zhu, S., and Wek, R.C. (1995). Mol. Cell. Biol. 15, 4497– 4506.