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 phosphorylati ng translation initiation factor 2, Several regulatory domains, includ ing a pseudokinase domain, a histidyl-tRNA synthetase (HisRS)-related region, and a C-terminal (C-term) segment required for ribosome associ ation, have been identified in GCN2, We used the yeast two-hybrid assa y, coimmunoprecipitation analysis, and in vitro binding assays to inve stigate 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 a nd the C-term domains could be coimmunoprecipitated with wild-type GCN 2 from yeast cell extracts, In addition, in vitro-translated PK and C- term segments showed specific binding in vitro to recombinant glutathi one S-transferase (GST)-PK and GST-C-term fusion proteins, respectivel y. Wild-type GCN2 could be coimmunoprecipitated with a full-length Lex A-GCN2 fusion protein from cell extracts, providing direct evidence fo r dimerization by full-length GCN2 molecules. Deleting the C-term or P K segments abolished or reduced, respectively, the yield of GCN2-LexA- GCN2 complexes. These results provide in vivo and in vitro evidence th at GCN2 dimerizes through self-interactions involving the C-term and P K domains, The PK domain showed pairwise in vitro binding interactions with the pseudokinase, HisRS, and C-term domains; additionally, the H isRS domain interacted with the C-term region, We propose that physica l interactions between the PK domain and its Banking regulatory region s and dimerization through the PK and C-term domains both play importa nt roles in restricting GCN2 kinase activity to amino acid-starved cel ls.