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Aminoacyl-tRNA synthetases (tRNA synthetases) of higher eukaryotes form a multiprotein complex. Sequence elements that are responsible for the protein assembly were searched by using a yeast two-hybrid system.Human cytoplasmic isoleucyl-tRNA synthetase is a component of the multi-tRNA synthetase complex and it contains a unique C-terminal appendix. This part of the protein was used as bait to identify an interacting protein from a HeLa cDNA library. The selected sequence represented the internal 317 amino acids of human bifunctional (glutamyl-and prolyl-) tRNA synthetase, which is also known to be a component of the complex. Both the C-terminal appendix of the isoleucyl-tRNA synthetase and the internal region of bifunctional tRNA synthetase comprise repeating sequence units, two repeats of about 90 amino acids, and three repeats of 57 amino acids, respectively. Each repeated motif of the two proteins was responsible for the interaction, but the stronger interaction was shown by the native structures containing multiple motifs.Interestingly, the N-terminal extension of human glycyl-tRNA synthetase containing a single motif homologous to those in the bifunctional tRNA synthetase also interacted with the C-terminal motif of the isoleucyl-tRNA synthetase although the enzyme is not a component of the complex. The data indicate that the multiplicity of the binding motif in the tRNA synthetases is necessary for enhancing the interaction strength and may be one of the determining factors for the tRNA synthetases to be involved in the formation of the multi-tRNA synthetase complex.Aminoacyl-tRNA synthetases play an essential role in cellular protein synthesis by catalyzing attachment of their cognate amino acids to tRNAs. Many eukaryotic aminoacyl-tRNA synthetases are distinguished from their prokaryotic counterpart in their abilities to form supracomplexes through self assembly or association with protein synthesis machinery (1-5) and cellular structures (6, 7). Most intriguing among them is a multi-tRNA synthetase complex generated by the assembly of many aminoacyl-tRNA synthetases and a few other protein factors of unknown function (8-10). The exact structure of the complex is still controversial because different forms of the complex have been isolated depending on the purification methods and organisms. The eukaryotic tRNA synthetases have been grouped depending on their abilities to form the multi-tRNA synthetase complex (8). The class I enzymes includes isoleucyl-, leucyl-, methionyl-, aspartyl-, bifunctional (glutamyl-and prolyl-), glutaminyl-, lysyl-, and arginyl-tRNA synthetases that have been consistently identified as the components of the multi-tRNA synthetase complex (11, 12). It is not clear whether other tRNA synthetases are loosely associated with or completely independent of such a complex.Understanding the structure of the multi-tRNA synthetase complex is important to find the functional linkage between the catalytic activities of this complex and other cellular processes. Many different approa...

Akt is stimulated by several growth factors and has a major anti-apoptotic role in the cell. Therefore, we hypothesized that a pathway leading to the inhibition of Akt might be utilized in the process of apoptosis. Accordingly, we used a yeast two-hybrid screening assay to identify the proteins that interact with and possibly inhibit Akt. We found that the C-terminal region of protein kinase C-related kinase 2 (PRK2), containing amino acids 862 to 908, specifically binds to Akt in yeast and mammalian cells. During early stages of apoptosis, the C-terminal region of PRK2 is cleaved from the inhibitory N-terminal region and can bind Akt. The proteinprotein interaction between Akt and the PRK2 C-terminal region specifically down-modulates the protein kinase activities of Akt by inhibiting phosphorylation at threonine 308 and serine 473 of Akt. This inhibition of Akt leads to the inhibition of the downstream signaling of Akt in vivo. The PRK2 C-terminal fragment strongly inhibits the Akt-mediated phosphorylation of BAD, a pro-apoptotic Bcl-2 family protein, and blocks the antiapoptotic activities of Akt in vivo. These results provide direct evidence that the products of protein cleavage during apoptosis inhibit pro-survival signalings, leading to the amplification of pro-apoptotic signalings in the cell.

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