title: Serine 577 is phosphorylated and negatively affects the tRNA binding and eIF2alpha kinase activities of GCN2.
abstract: Protein kinase GCN2 regulates translation initiation by phosphorylating eukaryotic initiation factor 2alpha (eIF2alpha), impeding general protein synthesis but specifically inducing translation of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. GCN2 activity is stimulated in amino acid-deprived cells through binding of uncharged tRNA to a domain related to histidyl tRNA synthetase. We show that GCN2 is phosphorylated by another kinase on serine 577, located N-terminal to the kinase domain. Mutation of Ser-577 to alanine produced partial activation of GCN2 in nonstarved cells, increasing the level of phosphorylated eIF2alpha, derepressing GCN4 expression, and elevating the cellular levels of tryptophan and histidine. The Ala-577 mutation also increased the tRNA binding affinity of purified GCN2, which can account for the elevated kinase activity of GCN2-S577A in nonstarved cells where uncharged tRNA levels are low. Whereas Ser-577 remains phosphorylated in amino acid-starved cells, its dephosphorylation could mediate GCN2 activation in other stress or starvation conditions by lowering the threshold of uncharged tRNA required to activate the protein.
authors.affiliation: Laboratory of Gene Regulation and Development, NICHD/National Institutes of Health, Building 6A, Rm. B1A13, Bethesda, MD 20892, USA.
authors.forename: Minerva
authors.initials: M
authors.lastname: Garcia-Barrio
authors.forename: Jinsheng
authors.initials: J
authors.lastname: Dong
authors.forename: Vera A
authors.initials: VA
authors.lastname: Cherkasova
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Zhang
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.forename: Sandra
authors.initials: S
authors.lastname: Ufano
authors.forename: Ruby
authors.initials: R
authors.lastname: Lai
authors.forename: Jun
authors.initials: J
authors.lastname: Qin
authors.forename: Alan G
authors.initials: AG
authors.lastname: Hinnebusch
mesh_terms: D013058:Mass Spectrometry; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D012343:RNA, Transfer / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship; D019892:eIF-2 Kinase / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D012694:Serine; D012343:RNA, Transfer; D011494:Protein Kinases; C512912:GCN2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase