Methylation of human elongation factor eEF1A2 is not essential for eEF1A2-eEF1B interaction

© 2020 L. V. Porubleva et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 577.112.7


Introduction
Translation elongation factor eEF1A*GTP carries aminoacyl-tRNA to the A-site of the 80S ribosome facilitating the process of ribosomal protein synthesis. GTP hydrolysis fina lizes the codon-anticodon recognition, then eEF1A*GDP leaves the ribosome [1]. The eEF1B complex comprising Bα, Bβ and Bγ subunits helps to exchange GDP for new GTP in eEF1A molecule [2,3], that is why the eEF1AeEF1B interaction is very important for translation to proceed.
Methylation of lysine residues is an important modification involved in the regulation of cell activity. For instance, methylation of specific lysine residues in histones indorses binding of the proteins and induces transcriptional silencing [17]. On the other hand, methylation of lysine residues in histones can impede binding of effector molecules [18]. Methylation of lysine residues has been found in several nonhistone proteins, including transcription factors, receptors, ribosomal proteins, and translation factors [19][20][21][22]. A significant regulatory potential of this modification is emphasized by the discovery of demethylases, the enzymes that can remove a methyl group or several methyl groups from the already modified proteins [23]. Recent studies indicate the involvement of methylation/demethylation processes in both histone and non-histone proteins into oncogenesis [24][25][26][27].
Methylation of five conservative lysine residues of eEF1A (K36, K55, K79, K165 and K318) is known for more than 20 years [28]. A role of eEF1A methylation in tumorigenesis was recently suggested [29], however, the molecular mechanisms involved are not clear. Since all lysine residues of eEF1A capable of methylation are situated outside the protein globule [30,31], we reasoned that methylation of these residues might affect the interaction of eEF1A with other proteins. eEF1B is one of the main translational partners of eEF1A during the elongation step of protein synthesis. Here, we study the effect of methylation of the protooncogenic eEF1A2 isoform on its interaction with eEF1B complex.
Cells 293 were grown at 37° C, 5 % CO 2 , 100 % humidity in DMEM containing 5 % FBS and antibiotics penicillin and streptomycin and glutamine. Transfection of 293 cells with plasmids pFC14K/eEF1A2, pFC14K/ eEF1A2(KxxR) and control vector HaloTag was performed with TurboFect reagent (Thermo Scientific, USA) for 24 h, after which cells were pelleted, lysed using mammalian lysis buffer (Promega, USA) at -80° C until use. Affinity purification of partner proteins was performed using the HaloTag® Complete Pull-Down System (Promega, USA) according to the manufacturer's instructions. Proteins were separated in 10 % PAGE for expression control of HaloTageEF1A2(KxxR). Proteins from lysates separated by SDS-PAGE were transferred to PVDF membrane (HybondP) at 30 V for 30 min. The membrane was then blocked for 1 h at room temperature in 5 % dried nonfat milk in Tris-buffered saline. After blocking, the membranes were incubated with primary antibodies (1:10000 rabbit anti-Halo-Tag, Promega, USA) in 3 % dried non-fat milk in 1 X Tris-buffered saline with 0.1 % Tween 20 (v/v, TBST) overnight at 4°C. After washing with 1X TBST, the membrane was incubated with anti-rabbit IgG-HRP (1:5000) se con dary antibody in 3 % dried nonfat milk in 1 X TBST or anti-mouse antibody (1:10000) in 3 % dried nonfat milk in 1 X TBST for 1 h at room temperature. ECL was used to visuali ze bands probed with HRP secondary antibody (Amersham Biosciences ECL Prime). The gel was photographed on a ChemiDoc tool (BioRad, USA). Densitometry was performed using ImageLab software (BioRad, USA).
The Control Vector plasmid (Promega, USA) expressing the HaloTag alone was used as a negative control. 300 000 сells 293 were plated into a well of a 12-well plate and were grown overnight in DMEM medium at 37°C, 5 % CO. After the attachment to wells the cells were transfected with TurboFect reagent (Thermo Scientific, USA) by plasmids 2 µg HaloTag plasmid -pFC14K/eEF1A2 or its mutants or HaloTag Control Vector and 0.02 µg NanoLuc Plasmid pNLF1/eEF1Bα (ratio 100 : 1). In 24 hours the cells were replanted into 96well plate (2 × 10 5 cells/well) in Opti-MEM ® I Reduced Serum Medium, no phenol red (Life Technologies, USA) + 4 % FBS, containing 100 nM HaloTag ® NanoBRET™ 618 Ligand or 0.1 % DMSO. Cells were іncubated at 37°C, 5 % CO 2 overnight (18-24 hours). 5X solution of NanoBRET™ Nano-Glo ® Substrate in Opti-MEM ® I Reduced Serum Medium without phenol red was added to each well up to 1 x final dilution immediately before measurement. Donor emission (460nm) and acceptor emission (618nm) were measured using plate reader Synergy HT (Bio-Tek Instrument, USA).

Results and Discussion
To investigate the role of methylation of several specific Lys residues in eEF1A2 we produced the mutant proteins where these residues were replaced by Arg. Five mutants with a single substitution, K36R, K55R, K79R, K165R or K318R, were generated. "Full" mutant (FM) was also produced, in which all five indicated Lys residues were replaced by Arg. Arg cannot be methylated by methyltrasferases specific for Lys and the replacement of Lys by Arg should not cause significant conformational changes in the protein, since Arg retains a positive charge of the Lys side chain. Here, the energy transfer and pull down approaches were applied to investigate the interaction of these mutants with different subunits of the eEF1B complex.
The mutant and wildtype eEF1A2 proteins were cloned into plasmid pFC14K enco ding HaloTag. The tag was located at the C-terminus of eEF1A2, since a majority of the Lys residues of interest were confined to the Nterminal part of the protein. In this case, HaloTag would not create steric hindrance to the interaction with the partner proteins. The resulting plasmid encoded eEF1A2HaloTag with a molecular weight of about 85 kDa (50 kDa eEF1A2 and 35 kDa HaloTag).
To estimate a possible effect of the eEF1A2 methylation on its interaction with eEF1B in intracellular environment we used a bioluminescence resonance energy transfer (BRET) system. The interaction between the two fusion proteins, one of which carries luminescent label and another contains fluorescent label, can make these labels close enough for the resonance energy transfer to occur. In this case, we used bioluminescent NanoLuc luciferase (Promega, USA) fused with eEF1Bα and a fluorescent ligand linked to HaloTag in eEF1A2.
293 cells were transfected with the plasmids encoding wildtype eEF1A2 and its mutants containing HaloTags and eEF1Bα fused with the NanoLuc domain. Fig. 1 shows that BRET between eEF1A2HaloTag and nanoLuc eEF1Bα is easily detected evidencing the existence of eEF1A2eEF1B interaction in 293 cells. Importantly, no methylationdeficient mutants showed a difference at the BRET level as compared to wildtype eEF1A2. It implies that methylation of the Lys residues in positions 36, 55, 79, 165 and 318 of eEF1A2 may have no significant impact on its interaction with the eEF1B complex in human cells.
However, it remains possible that we could not detect the methylation effect due to some limitations of the BRET method, which provides measurements directly in cells. Additionally, we used only one subunit of eEF1B for BRET experiments. So, we employed the HaloTag-based pull down procedure to test if we could observe a difference in the wild type and mutant eEF1A2 proteins binding to diffe rent subunits of eEF1B in cell lysates by Western blotting.
The HaloTag® Pull-Down system was used for pull down experiments. The amount of Halo label in the lysates of cells expressing different proteins with HaloTag was estimated by Western blotting with anti-HaloTag antibodies. Then the HaloTag affinity purification system for protein complexes was used to pull down the protein partners of eEF1A2. The proteins were separated by polyacrylamide gel electrophoresis. Noteworthy, before starting the pull down procedure the volume of each electrophoresis sample was adjusted to correspond to the normalized amount of eEF1A HaloTag, measured by Western blot of the cell lysates with antiHaloTag antibodies (Fig. 2). Besides, to control the level of binding of HaloTag protein to the resin, the portions of cell lysates after exhausting by pull down were loaded on the same gel. This was necessary to ensure the correct comparison of the protein levels in each sample. Western blots with the antibodies recognizing eEF1Bα, eEF1Bβ and eEF1Bγ subunits were subjected to densito metry to quantitate the subunits levels in the eluates of the pulled down proteins.
Densitometry analysis of the blots confirmed the interaction of eEF1B subunits with eEF1A2 (Fig. 3). It should be kept in mind that eEF1A is capable to interact directly with the eEF1Bα and eEF1Bβ subunits whereas an apparent interaction of eEF1A with eEF1Bγ, observed on Western blots, in fact just reflects  eEF1A binding to other subunits of the eEF1B complex [34]. Thus, in the case of eEF1Bγ, we actually observed the interaction of eEF1A2 with the eEF1B complex. Again, no essential changes in the amount of the eEF1Bα, eEF1Bβ and eEF1Bγ subunits pulled down by the methylation-free mutants were detected as compared to wildtype eEF1A2 (Fig. 2). The mutant K165R was not studied in this case, as К165 methylation did not influence the interactions of eEF1A with other elongation factors in pull down procedure [35]. Thus, methylation of eEF1A2 apparently has no substantial effect on its interaction with the eEF1B complex both in vitro and in cellular.
After identification of five methyltransferases of eEF1A that are unique for every lysine residue involved [29,[36][37][38] it became clear that eEF1A is the only known so far target of these enzymes. It is known that methylation of K55 in eEF1A may be important for tumorigenesis [29,39] but a potential mechanism of the impact of K55 as well as of K36, K79, K165 and K318 methylation remains to be elucidated. As the methylated Lys residues are localized on the surface of the eEF1A proteins one may suggest that this modification may influence the interaction of eEF1A with the protein partners. eEF1A has a number of confirmed translational [40,41] and nontranslational [7,8,[42][43][44] protein partners, thus the search for methylation-impacted protein-protein contacts is at the initial stage.
Our data suggest a removal of the altered eEF1A2eEF1B interaction from the list of potential molecular mechanisms explaining how the methylation influences a tumorigenic action of eEF1A2. As far as translation is concerned, methylation of eEF1A2 may be still important for its interaction with ribosomal proteins [45,46] and/or aminoacyltRNA synthetases [47]. Besides, eEF1A2 fulfills a number of moonlighting functions [48], so methylation may have an impact on the eEF1A2 interaction with different non-translational proteins in cancer cells.
This work was supported in part by the budget program KPKVK 6541230, pro ject №0120U100648.