Identification of positive-acting domains in GCN2 protein kinase required for translational activation of GCN4 expression.
GCN4 is a transcriptional activator of amino acid-biosynthetic genes in the yeast Saccharomyces cerevisiae. GCN2, a translational activator of GCN4 expression, contains a domain homologous to the catalytic subunit of eucaryotic protein kinases. Substitution of a highly conserved lysine residue in the kinase domain abolished GCN2 regulatory function in vivo and its ability to autophosphorylate in vitro, indicating that GCN2 acts as a protein kinase in stimulating GCN4 expression. Elevated GCN2 gene dosage led to derepression of GCN4 under nonstarvation conditions; however, we found that GCN2 mRNA and protein levels did not increase in wild-type cells in response to amino acid starvation. Therefore, it appears that GCN2 protein kinase function is stimulated posttranslationally in amino acid-starved cells. Three dominant-constitutive GCN2 point mutations were isolated that led to derepressed GCN4 expression under nonstarvation conditions. Two of the GCN2(Con) mutations mapped in the kinase domain itself. The third mapped just downstream from a carboxyl-terminal segment homologous to histidyl-tRNA synthetase (HisRS), which we suggested might function to detect uncharged tRNA in amino acid-starved cells and activate the adjacent protein kinase moiety. Deletions and substitutions in the HisRS-related sequences and in the carboxyl-terminal segment in which one of the GCN2(Con) mutation mapped abolished GCN2 positive regulatory function in vivo without lowering autophosphorylation activity in vitro. These results suggest that sequences flanking the GCN2 protein kinase moiety are positive-acting domains required to increase recognition of physiological substrates or lower the requirement for uncharged tRNA to activate kinase activity under conditions of amino acid starvation.
Unit on Molecular Genetics of Lower Eukaryotes, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
R C
RC
Wek
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M
Ramirez
B M
BM
Jackson
A G
AG
Hinnebusch
D000483:Alleles; D000936:Antigen-Antibody Complex / Q000032:analysis; D003062:Codon / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005799:Genes, Dominant; D005800:Genes, Fungal; D005809:Genes, Regulator; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic
D000936:Antigen-Antibody Complex; D003062:Codon; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011494:Protein Kinases