Dual role of methionyl-tRNA synthetase in the regulation of translation and tumor suppressor activity of aminoacyl-tRNA synthetase-interacting multifunctional protein-3

Abstract

Mammalian methionyl-tRNA synthetase (MRS) plays an essential role in initiating translation by transferring Met to initiator tRNA (tRNA(i)(Met)). MRS also provides a cytosolic anchoring site for aminoacyl-tRNA synthetase-interacting multifunctional protein-3 (AIMP3)/p18, a potent tumor suppressor that is translocated to the nucleus for DNA repair upon DNA damage. However, the mechanism by which this enzyme mediates these two seemingly unrelated functions is unknown. Here we demonstrate that AIMP3 is released from MRS by UV irradiation-induced stress. Dissociation was induced by phosphorylation of MRS at Ser662 by general control nonrepressed-2 (GCN2) following UV irradiation. Substitution of Ser662 to Asp (S662D) induced a conformational change in MRS and significantly reduced its interaction with AIMP3. This mutant possessed significantly reduced MRS catalytic activity because of loss of tRNA(Met) binding, resulting in down-regulation of global translation. According to the Met incorporation assay using stable HeLa cells expressing MRS S662A or eukaryotic initiation factor-2 subunit-α (eIF2α) S51A, inactivation of GCN2-induced phosphorylation at eIF2α or MRS augmented the role of the other, suggesting a cross-talk between MRS and eIF2α for efficient translational inhibition. This work reveals a unique mode of regulation of global translation as mediated by aminoacyl-tRNA synthetase, specifically MRS, which we herein identified as a previously unidentified GCN2 substrate. In addition, our research suggests a dual role for MRS: (i) as a coregulator with eIF2α for GCN2-mediated translational inhibition; and (ii) as a coupler of translational inhibition and DNA repair following DNA damage by releasing bound tumor suppressor AIMP3 for its nuclear translocation.

Fig. 1.

Schematic representation of functional domain arrangements in human MRS and AIMP3. Human MRS contains an N-terminal extension that has homology to GST (gray). The central catalytic domain (white) contains class I signature motifs, such as HIGH and KMSKS. The C-terminal domain (pink for anticodon binding and green for WHEP domains) interacts with tRNA^Met. The GST-homology domain of MRS specifically interacts with another GST-homology domain (gray) of AIMP3. Ser662 is phosphorylated by UV-activated GCN2. D1 (1–266 residues), D2 (267–597), and D3 (598–900) are the deletion fragments of MRS that were used for further assays.

Fig. 2.

AIMP3 dissociates from MRS and translocates to the nucleus following UV irradiation. (A) MRS-interacting proteins in UV-irradiated HeLa cells (60 J/m²) were determined by IP. Levels of proteins in whole cell lysates (WCL) were also detected by immunoblotting. (B) HCT116 cells cotransfected with Flag–AIMP3-VN and HA–MRS-VC were UV-irradiated. Reconstitution of Venus fluorescence by the interaction of MRS and AIMP3 is shown by green fluorescence. AIMP3 alone was observed by red fluorescence of the Alexa Fluor 555-conjugated anti-Flag Ab (200x). (C) The graph presents the relative ratios of green (MRS:AIMP3 complex) versus red (AIMP3) spot counts. Data are represented as mean ± SD (n = 3). (D) The dissociation of AIMP3 from MRS and nuclear localization was monitored at higher magnification (600x) using HeLa cells. The red fluorescence from AIMP3 merged with DAPI-stained nucleus, forming foci. (E) After UV irradiation, cytosolic and nuclear AIMP3 was analyzed by immunoblotting. YY1 and tubulin were used as controls.

Fig. 3.

GCN2-induced phosphorylation is responsible for the dissociation of MRS and AIMP3. (A and B) HeLa cells treated with si-control or si-GCN2 for 72 h (A) or GCN2^+/+ and GCN2^−/− MEFs (B) were UV-irradiated. Interaction between MRS and AIMP3 was detected by IP and immunoblotting. The relative ratio of bound AIMP3 to MRS was quantified and presented to show the extent of AIMP3 dissociation. (C) Proteins from HeLa cells were separated by 2D-PAGE. Half of the UV-irradiated samples were treated with alkaline phosphatase (AP) for dephosphorylation. (D) The effect of GCN2 knockdown on MRS phosphorylation was detected (Upper). UV-dependent phosphorylation of GCN2 was validated using p-Thr Ab. (E) Cellular interaction of GCN2 with MRS was observed in UV-irradiated HeLa cells. (F) Kinase assay was done by incubation of Flag-GCN2, [γ-³²P]ATP, and MBP-MRS D1, D2, and D3. After stained with Coomassie brilliant blue (CBB), gel was dried and exposed for autoradiography. (G) The immobilized GST-GCN2 kinase domain (KD) was mixed with biotinylated synthetic peptides containing known sequences of GCN2 substrates (GCN2 Thr899 and eIF2α Ser51) and [γ-³²P]ATP to detect phosphorylation. An inactive form of GCN2 KD K618R mutant was used as a control.

Fig. 4.

GCN2-induced phosphorylation may trigger a conformational change in MRS and induce AIMP3 dissociation. (A) Radioactively labeled AIMP3 was incubated with immobilized GST-MRS WT and MRS mutants. (B) MRS WT or the S662D mutant was coexpressed with His-AIMP3 using a dual protein expression system, and the bound protein to AIMP3 was detected by immunoblotting. (C) The interaction of MRS WT and the S662D mutant with AIMP3 by UV was determined by IP using HeLa cells stably expressing Myc-tagged MRS proteins. (D) CD spectra of the purified MBP-MRS WT and S662D mutant were obtained in the near UV. (E) Purified MBP-MRS proteins were digested with trypsin or elastase, and the cleaved fragments were visualized by CBB staining. (F) Peptide linker was inserted between the residues 233 and 234 of MRS WT and of the S662D mutant. The native and peptide linker-inserted MRS proteins were subjected to an in vitro pull-down assay with radioactively labeled AIMP3.

Fig. 5.

The effect of MRS phosphorylation on tRNA binding and global translation. (A and B) The tRNA_i^Met probe was incubated with His-MRS WT and mutant proteins and separated by nondenaturing PAGE (A) or filtered through a nitrocellulose membrane (B). (C) Relative catalytic activities of His-MRS WT and mutants were assessed by aminoacylation assay. Data are represented as mean ± SD (n = 3). (D) HeLa cells were incubated with [³⁵S]Met with or without UV irradiation. Total RNA was extracted and charged tRNA^Met was detected by autoradiography (Left). The relative Met-tRNA^Met amount was quantified by digital imaging (Center). The values are represented as mean ± SD (n = 2). Protein synthesis under the same conditions was monitored by the [³⁵S]Met incorporation assay (Right). Data are represented as mean ± SD (n = 3). (E) Endogenous MRS in stable HeLa cells expressing Myc-tagged MRS WT were knocked down by siRNA. (F) Endogenous MRS was down-regulated in MRS WT and S662D stable HeLa cells (Left) and stable cell clones were assayed for global protein synthesis. We chose stable cells highly expressing the S662D mutant to see its overexpressional effect on translation. Data are represented as mean ± SD (n = 3) (Right).

Fig. 6.

MRS coordinates with eIF2α to regulate translation upon UV irradiation. (A and B) HeLa cells treated with si-eIF2α or si-MRS were UV-irradiated and p-MRS and p-eIF2α was monitored. (C and D) UV-induced phosphorylation of MRS and eIF2α was monitored in stable cell lines expressing the eIF2α S51A or MRS S662A mutant. Empty vector (EV)-transfected cells were used as a control (C). The Myc-tagged eIF2α S51A mutant migrated slowly and appeared in the upper band of endogenous eIF2α. (E) Protein synthesis after UV irradiation was measured in stable cells expressing MRS WT and the S662A mutant (Left) and EV and the eIF2α S51A mutant (Right). Endogenous MRS in cells expressing MRS WT and the S662A mutant was knocked down by siRNA treatment.

Fig. 7.

Schematic representation for the dual role of MRS. Under normal condition, MRS catalyzes aminoacylation of tRNA^Met for translational initiation. AIMP3 is mainly bound to the N-terminal extension of MRS. Upon UV irradiation, MRS is phosphorylated at Ser662 by GCN2, and binding of tRNA^Met was blocked. Phosphorylation of MRS induces a conformational change, causing the dissociation of AIMP3 from MRS. Released AIMP3 is translocated to the nucleus to mediate DNA damage repair. GCN2 also phosphorylates eIF2α and p-MRS and p-eIF2α work together to control global translation.