Precursor of Pro-apoptotic Cytokine Modulates Aminoacylation Activity of tRNA Synthetase

Abstract

Endothelial monocyte activating polypeptide II (EMA-PII) is a cytokine that is specifically induced by apoptosis.Its precursor (pro-EMAPII) has been suggested to be identical to p43, which is associated with the multi-tRNA synthetase complex. Herein, we have demonstrated that the N-terminal domain of pro-EMAPII interacts with the Nterminal extension of human cytoplasmic arginyl-tRNA synthetase (RRS) using genetic and immunoprecipitation analyses. Aminoacylation activity of RRS was enhanced about 2.5-fold by the interaction with pro-EMAPII but not with its N-or C-terminal domains alone. The N-terminal extension of RRS was not required for enzyme activity but did mediate activity stimulation by pro-EMAPII. Pro-EMAPII reduced the apparent K m of RRS to tRNA, whereas the k cat value remained unchanged. Therefore, the precursor of EMAPII is a multi-functional protein that assists aminoacylation in normal cells and releases the functional cytokine upon apoptosis.Aminoacyl-tRNA synthetases (ARSs) 1 catalyze ligation of their cognate amino acids to specific tRNAs. Although basic architecture of the core domain is well conserved among ARSs, unique peptide extensions have been found in the N-or Cterminal ends of metazoan enzymes (1-3). Although these extensions have been thought to be involved in heterologous molecular interactions, their functional significance is not yet understood.A macromolecular protein complex consisting of at least nine different ARSs has been found in higher eukaryotes (1-3). This multi-ARS complex also contains three nonsynthetase components, p18, p38, and p43 whose functions are not clear (4 -7). Among these nonsynthetase components, p43 has been proposed to be a precursor of a tumor-specific cytokine, endothelial monocyte-activating polypeptide II (EMAPII) based on over 80% sequence identity between the two proteins (6). EMAPII was originally identified in the culture medium of murine fibrosarcoma cells induced by methylcholanthrene A (8). It triggers an acute inflammatory response (9, 10) and is involved in development-related apoptosis (11).The precursor for EMAPII (pro-EMAPII) is processed at the Asp residue of ASTD/S sequence to release the C-terminal cytokine domain of 23 kDa (11). Its C-terminal domain shares homology with the C-terminal parts of methionyl-tRNA synthetases of prokaryotes, archaea and nematode, and also a yeast protein, Arc1p/G4p, which interacts with methionyl-and glutamyl-tRNA synthetases. The N-terminal domain of pro-EMAPII does not show homology to any known proteins, and its function has not been understood.EMAPII is expressed in a wide range of cell lines and normal tissues (12) and its mRNA level is unchanged during apoptosis (11) although its production is induced by apoptosis. The present work was designed to address whether pro-EMAPII is identical to p43 and to understand its function in the normal cell. The results showed that pro-EMAPII is associated with the N-terminal extension of human arginyl-tRNA synthetase (RRS), facilitating aminoacylation reaction. EXPE...

A Three-dimensional Working Model of the Multienzyme Complex of Aminoacyl-tRNA Synthetases Based on Electron Microscopic Placements of tRNA and Proteins

“…By similar reasoning, the combination of cross-linking and protein interaction studies suggests that one upper domain of the structure contains ArgRS and LysRS and so the remaining enzymes are in the other domain. The auxiliary protein p43 has been shown to interact with ArgRS (11) and is near the center of the structure (12). Thus, the general location of tRNA Arg is likely to be near area 3, which may also include tRNA Lys .…”

“…Evidence that this is appropriate comes from data showing that ArgRS is stabilized within the complex by the N-terminal portion of p43 (10). In addition, it is thought that a major function of complex-associated p43, a known tRNA-binding protein, is to facilitate enzymatic activities within the complex, in particular ArgRS (11). However, when working with individual purified proteins (10) no catalytic enhancement of ArgRS due to association with p43 was seen.…”

Glutamine-dependent Antiapoptotic Interaction of Human Glutaminyl-tRNA Synthetase with Apoptosis Signal-regulating Kinase 1

“…Rabbit polyclonal antibody was then raised against these proteins as described previously (20). The IgG from antiserum was purified by protein A affinity chromatography according to the manufacturer's protocol (Bio-Rad).…”

“…Thus, tyrosyl-tRNA synthetase has a potent apoptosis-inducing activity after proteolytic cleavage, since their split polypepetides stimulate the production of TNF and tissue factor from target cells and have leukocyte chemotaxis activity to scavenge apoptotic corpses. The precursor of endothelial monocyte-activating polypeptide II is associated with the N-terminal noncatalytic extension of arginyl-tRNA synthetase and enhances aminoacylation activity (20). Like human tyrosyl-tRNA synthetase, the cytokine domain of this precursor is released upon apoptosis and exerts its proapoptotic function (21,22).…”

AIMP2/p38, the scaffold for the multi-tRNA synthetase complex, responds to genotoxic stresses via p53

“…Many of these complex-forming ARSs, as well as AIMPs, play diverse regulatory roles that are not directly related to protein synthesis (2). Among the three AIMPs, AIMP1 is secreted as a cytokine working in immune, angiogenesis, and wound-healing processes (3)(4)(5)(6)(7) and also functions as a hormone controlling glucose homeostasis (8). AIMP3 is a tumor suppressor required for chromosome integrity (9,10).…”