Noncanonical Function of Glutamyl-Prolyl-tRNA Synthetase

Sampath, Prabha; Mazumder, Barsanjit; Seshadri, Vasudevan; Gerber, Carri A.; Chavatte, Laurent; Kinter, Michael; Ting, Shu-mei; Dignam, John David; Kim, Sung Hoon; Driscoll, Donna M.; Fox, Paul L.

doi:10.1016/j.cell.2004.09.030

Cited by 226 publications

(292 citation statements)

References 55 publications

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“…Although levels of FBP and c-Myc in AIMP2-deficient cells are actually higher than in WT cells (12), the level of p53 and its activity are more suppressed, implying that the pathway of AIMP2 to p53 would not be directly linked to the AIMP2-FBP-c-Myc pathway in response to TGF-␤ or the up-regulation of c-Myc in response to UV irradiation (27). EPRS was previously shown to be phosphorylated and dissociated from the complex by IFN-␥ treatment to form a new multiprotein complex that suppresses translation of specific transcripts (28). However, EPRS does not seem to be mobilized by UV irradiation (Fig.…”

Section: Discussionmentioning

confidence: 99%

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

Han¹,

Park²,

Park³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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107

108

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AIMP2/p38 is a scaffolding protein required for the assembly of the macromolecular tRNA synthetase complex. Here, we describe a previously unknown function for AIMP2 as a positive regulator of p53 in response to genotoxic stresses. Depletion of AIMP2 increased resistance to DNA damage-induced apoptosis, and introduction of AIMP2 into AIMP2-deficient cells restored the susceptibility to apoptosis. Upon DNA damage, AIMP2 was phosphorylated, dissociated from the multi-tRNA synthetase complex, and translocated into the nuclei of cells. AIMP2 directly interacts with p53, thereby preventing MDM2-mediated ubiquitination and degradation of p53. Mutations in AIMP2, affecting its interaction with p53, hampered its ability to activate p53. Nutlin-3 recovered the level of p53 and the susceptibility to UV-induced cell death in AIMP2-deficient cells. This work demonstrates that AIMP2, a component of the translational machinery, functions as proapoptotic factor via p53 in response to DNA damage.A minoacyl-tRNA synthetases (ARSs) are the enzymes that ligate specific amino acids to tRNAs before protein synthesis. In higher eukaryotic systems, nine different ARSs form an intriguing macromolecular complex with three nonenzymatic factors called ARS-interacting, multifunctional proteins (AIMPs) (1, 2). 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 (3-7) and also functions as a hormone controlling glucose homeostasis (8). AIMP3 is a tumor suppressor required for chromosome integrity (9, 10). Although AIMP2 is critical for the assembly of the multi-ARS complex (11), it also suppresses cell proliferation via down-regulation of c-Myc (12). In addition, AIMP2 was shown to be involved in Parkinson's disease, inducing neural cell death (13). However, it is yet to be determined how AIMP2 is involved in the control of cell death. In this work, we investigated the functional significance and molecular behavior of AIMP2 during the control of cell death and the relationship of AIMP2 associated with the multi-ARS complex and its proapoptotic activity. Results AIMP2-Deficient Cells AreResistant to Cell Death. To see the importance of AIMP2 during the control of cell death, we subjected 12.5-d AIMP2 ϩ/ϩ and AIMP2 Ϫ/Ϫ mouse embryonic fibroblasts (MEFs) to UV irradiation and compared their apoptotic sensitivity. The apoptotic cells, indicated by the subG1 portion, were increased Ϸ3-fold by UV irradiation in AIMP2 ϩ/ϩ but not in AIMP2 Ϫ/Ϫ cells (Fig. 1A). Transfection of AIMP2 into AIMP2 Ϫ/Ϫ MEFs restored the apoptotic sensitivity to UV irradiation (Fig. 1B). We also compared the apoptotic response of AIMP2 ϩ/ϩ and AIMP2 Ϫ/Ϫ MEFs to UV irradiation by monitoring caspase-3 activation. Procaspase-3 cleavage resulting in caspase-3 generation was observed in AIMP2 ϩ/ϩ but not in AIMP2 Ϫ/Ϫ cells (Fig. 1C). Suppression of AIMP2 via gene-specific siRNA...

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Section: Discussionmentioning

confidence: 99%

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

Han¹,

Park²,

Park³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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107

108

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“…If this is the case, the binding of AIMP3 to its two target proteins, MRS and ATM, might be controlled by post-translational modification. In this regard, it should be noted that glutamylprolyl-tRNA synthetase, the largest component of the multi-ARS complex, is phosphorylated and dissociates from the complex after interferon-␥ treatment to form some different type of multi-protein complex that is required for gene silencing (39). Thus, it is possible that AIMP3 binding and movement could also be controlled by phosphorylation or some other post-translational modification.…”

Section: Discussionmentioning

confidence: 99%

Determination of Three-dimensional Structure and Residues of the Novel Tumor Suppressor AIMP3/p18 Required for the Interaction with ATM

Kim

Park

Choi

et al. 2008

Journal of Biological Chemistry

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Although AIMP3/p18 is normally associated with the multitRNA synthetase complex via its specific interaction with methionyl-tRNA synthetase, it also works as a tumor suppressor by interacting with ATM, the upstream kinase of p53. To understand the molecular interactions of AIMP3 and the mechanisms involved, we determined the crystal structure of AIMP3 at 2.0-Å resolution and identified its potential sites of interaction with ATM. AIMP3 contains two distinct domains linked by a 7-amino acid (Lys 57 -Ser 63 ) peptide, which contains a 3 10 helix. The 56-amino acid N-terminal domain consists of two helices into which three antiparallel ␤ strands are inserted, and the 111-amino acid C-terminal domain contains a bundle of five helices (Thr 64 -Tyr 152 ) followed by a coiled region (Pro 153 -Leu 169 ). Structural analyses revealed homologous proteins such as yeast glutamyl-tRNA synthetase, Arc1p, EF1B␥, and glutathione S-transferase and suggested two potential molecular binding sites. Moreover, mutations at the C-terminal putative binding site abolished the interaction between AIMP3 and ATM and the ability of AIMP3 to activate p53. Thus, this work identified the two potential molecular interaction sites of AIMP3 and determined the residues critical for its tumor-suppressive activity through the interaction with ATM.Although aminoacyl-tRNA synthetases (ARSs) 4 catalyze the ligation of specific amino acids to their cognate tRNAs, they are multifunctional proteins that are involved in the regulation of diverse signal pathways (1). Several ARSs of higher eukaryotes form an intriguing macromolecular protein complex with three non-enzymatic factors, designated AIMPs (ARS-interacting multifunctional proteins), which are also multiply involved in various biological processes. Of these factors, AIMP1/p43 is secreted as a cytokine that functions in immune response (2, 3), angiogenesis (4), and wound-healing processes (5) and also as a hormone that regulates glucose metabolism (6). AIMP2/p38 has been shown to mediate transforming growth factor-␤ signaling for c-Myc down-regulation (7). AIMP3/p18 binds to the multi-ARS complex by specifically interacting with methionyltRNA synthetase (MRS) and is translocated to the nucleus to activate p53 through the interaction with kinases such as ATM (ataxia telangiectasia mutated) and ATR (ATM-and Rad3-related) in response to DNA damage (8) or oncogenic stress (9). To understand the multifunctionality and regulation of the components of the multi-ARS complex, it is important to determine the three-dimensional structures of the components as well as the whole complex. So far, only the three-dimensional structures of the partial domains of the complex-forming ARSs such as glutamylprolyl-tRNA synthetase (10, 11), aspartyltRNA synthetase (12), and AIMP1/p43 (13, 14) have been elucidated. Here, we report the crystal structure of full-length AIMP3 and the residues and location required for the interaction with ATM. EXPERIMENTAL PROCEDURESCloning, Expression, and Purification of Human AIMP3-The cDN...

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“…6A), suggesting that the A/A system can be used to synthesize a wide array of proteins. The synthesis of the 163-kDa glutamyl-prolyl-tRNA synthetase (Sampath et al 2004) was also examined. Although we observed synthesis of the full-length protein, smaller products were also observed (data not shown).…”

Section: Extracts From A/a Mefs Synthesize Diverse Proteins In Large mentioning

confidence: 99%

An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation

Zeenko

Wang

Majumder

et al. 2008

RNA

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In vitro translation systems are used to investigate translational mechanisms and to synthesize proteins for characterization. Most available mammalian cell-free systems have reduced efficiency due to decreased translation initiation caused by phosphorylation of the initiation factor eIF2a on Ser51. We describe here a novel cell-free protein synthesis system using extracts from cultured mouse embryonic fibroblasts that are homozygous for the Ser51 to-Ala mutation in eIF2a (A/A cells). The translation efficiency of a capped and polyadenylated firefly luciferase mRNA in A/A cell extracts was 30-fold higher than in wild-type extracts. Protein synthesis in extracts from A/A cells was active for at least 2 h and generated up to 20 mg/mL of luciferase protein. Additionally, the A/A cell-free system faithfully recapitulated the selectivity of in vivo translation for mRNA features; translation was stimulated by a 59-end cap (m 7 GpppN) and a 39-end poly(A) tail in a synergistic manner. The system also showed similar efficiencies of cap-dependent and IRES-mediated translation (EMCV IRES). Significantly, the A/A cell-free system supported the post-translational modification of proteins, as shown by glycosylation of the HIV type-1 gp120 and cleavage of the signal peptide from b-lactamase. We propose that cell-free systems from A/A cells can be a useful tool for investigating mechanisms of mammalian mRNA translation and for the production of recombinant proteins for molecular studies. In addition, cell-free systems from differentiated cells with the Ser51Ala mutation should provide a means for investigating cell type-specific features of protein synthesis.

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Noncanonical Function of Glutamyl-Prolyl-tRNA Synthetase

Cited by 226 publications

References 55 publications

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

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

Determination of Three-dimensional Structure and Residues of the Novel Tumor Suppressor AIMP3/p18 Required for the Interaction with ATM

An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation

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