Dimerization by translation initiation factor 2 kinase GCN2 is mediated by interactions in the C-terminal ribosome-binding region and the protein kinase domain

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 Banking 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.