title: Dimerization by translation initiation factor 2 kinase GCN2 is mediated by interactions in the C-terminal ribosome-binding region and the protein kinase domain.
abstract: The protein kinase GCN2 stimulates translation of the transcriptional activator GCN4 in yeast cells starved for amino acids by phosphorylating translation initiation factor 2. Several regulatory domains, including a pseudokinase domain, a histidyl-tRNA synthetase (HisRS)-related region, and a C-terminal (C-term) segment required for ribosome association, have been identified in GCN2. We used the yeast two-hybrid assay, coimmunoprecipitation analysis, and in vitro binding assays to investigate physical interactions between the different functional domains of GCN2. A segment containing about two thirds of the protein kinase (PK) catalytic domain and another containing the C-term region of GCN2 interacted with themselves in the two-hybrid assay, and both the PK and the C-term domains could be coimmunoprecipitated with wild-type GCN2 from yeast cell extracts. In addition, in vitro-translated PK and C-term segments showed specific binding in vitro to recombinant glutathione S-transferase (GST)-PK and GST-C-term fusion proteins, respectively. Wild-type GCN2 could be coimmunoprecipitated with a full-length LexA-GCN2 fusion protein from cell extracts, providing direct evidence for dimerization by full-length GCN2 molecules. Deleting the C-term or PK segments abolished or reduced, respectively, the yield of GCN2-LexA-GCN2 complexes. These results provide in vivo and in vitro evidence that GCN2 dimerizes through self-interactions involving the C-term and PK domains. The PK domain showed pairwise in vitro binding interactions with the pseudokinase, HisRS, and C-term domains; additionally, the HisRS domain interacted with the C-term region. We propose that physical interactions between the PK domain and its flanking regulatory regions and dimerization through the PK and C-term domains both play important roles in restricting GCN2 kinase activity to amino acid-starved cells.
authors.affiliation: Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892-2716, USA.
authors.forename: H
authors.initials: H
authors.lastname: Qiu
authors.forename: M T
authors.initials: MT
authors.lastname: Garcia-Barrio
authors.forename: A G
authors.initials: AG
authors.lastname: Hinnebusch
mesh_terms: D001665:Binding Sites; D004268:DNA-Binding Proteins; D019281:Dimerization; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion
chemical_list: D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases