**2. Expression and interaction of eEF1A1 and eEF1A2 in HEK 293 cells**

To assess the possible physiological interaction between eEF1A isoforms in a natural cellular environment, such as the cytoplasm of intact cells, human embryonic kidney 293 (HEK 293) cell line was used as an experimental system. This choice was derived from the finding that HEK 293 cells normally express substantial levels of eEF1A1 isoform, whereas the eEF1A2 isoform is absent.

## **2.1. Expression in HEK 293 of eEF1A1 and eEF1A2**

**1. Introduction**

68 Protein-Protein Interaction Assays

Eukaryotic elongation factor 1A (eEF1A) belongs to the family of GTP-binding proteins and it is the second most abundant protein in the cellular environment. It catalyzes the first step of the elongation cycle by promoting the GTP-dependent binding of aminoacyl-tRNA to the A-site of the ribosome [1–3]. eEF1A exists as two isoforms eEF1A1 and eEF1A2 [4], and in humans, they share almost identical amino acid sequences (92% sequence identity). eEF1A1 is ubiquitously present except in skeletal and cardiac muscle, while eEF1A2 expression is restricted in the brain, skeleton muscle, heart, and other cell types including large motor neurons, islet cells in the pancreas, and neuroendocrine cells in the gut [5], and it is currently found in all vertebrates [6]. Besides their role in polypeptide synthesis, paralogous human eEF1A1 and eEF1A2 act as "moonlighting" proteins [7] owing to several noncanonical functions such as cytoskeleton remodeling by binding and bundling filamentous actin [8, 9], apoptosis, nuclear transport, proteasome-mediated degradation of damaged proteins, heat shock, and transformation [10–12]. Overexpression of eEF1A1 or eEF1A2 in Hela cells led to increased cell growth [7], whereas the disruption of eEF1A1 resulted in actin cytoskeleton defects under basal conditions and in response to palmitate, thus suggesting that eEF1A1 mediates lipotoxic cell death, secondary to oxidative and ER stress, by regulating cytoskeletal changes critical for this process [13]. These findings highlighted that eEF1A1 was involved in both cell proliferation and apoptosis, though the relationship between eEF1A1 and apoptosis is still unclear. By contrast, eEF1A2 seems to play antiapoptotic properties in ovarian, breast, pancreatic, liver, and lung cancer [14]; however, this oncogenic potential deserves further investigation [15].

The possible interaction between eEF1A molecules was first characterized in *Tetrahymena* as eEF1A dimer was able to bundle actin filament [16]. Subsequently, the identification of dimeric eEF1A was also reported in both chicken and human B cell lines [17]. Recent investigations indicated that, compared to eEF1A2, eEF1A1 showed a higher property of self-association [18]. Moreover, under oxidant condition, eEF1A1 was able to form intermolecular disulfide bonds [19]. Recent findings showed that C-Raf kinase interacts *in vivo* with eEF1A during a survival response mediated by epidermal growth factor (EGF) following the treatment of human lung cancer cells with α-interferon (IFNα) [20]. Moreover, phosphorylation of *e*EF1A *in vitro* by C-Raf on S21 required the presence of both *e*EF1A isoforms, thus suggesting that the existence of an eEF1A1/eEF1A2 complex and the S21 phosphorylation represented a regulatory mechanism responsible for the switch from eEF1A canonical to noncanonic functions [21]. On the basis of these findings, we recently showed the possible direct interaction between the *e*EF1A isoforms by using fluorescence resonance energy transfer (FRET) [22]. Compared to our previous work, here we settled for a different experimental approach mainly based on pull-down, confocal microscopy, and FRET analysis based on IgG-FITC (donor)- and IgG-TRITC (acceptor)-conjugated antibodies in HEK 293 cells transfected with recombinant His-tagged eEF1A2 isoform.

**2. Expression and interaction of eEF1A1 and eEF1A2 in HEK 293 cells**

To assess the possible physiological interaction between eEF1A isoforms in a natural cellular environment, such as the cytoplasm of intact cells, human embryonic kidney 293 (HEK 293) First, the efficiency of pcDNA3.1-eEF1A2(His)<sup>6</sup> (gift from C.R. Knudsen, Aarhus, Denmark [23]) to transfect HEK 293 cells was evaluated. As reported in **Figure 1A**, compared to non-transfected HEK 293, cells transfected with recombinant eEF1A2 isoform showed an increase in the expression of the 54 kDa bands corresponding to the molecular weight of eEF1A. Subsequently, the expression level of eEF1A2 using a specific anti-eEF1A2 antibody (prepared as already reported [22]) was analyzed. As shown in **Figure 1B**, eEF1A2 isoform was revealed only in HEK 293 cells transfected with pcDNA3.1-eEF1A2(His)<sup>6</sup> and confirmed with the anti-His antibody (Merck, Germany) (**Figure 1C**).

**Figure 1.** Expression of eEF1A isoforms in HEK 293 cells. HEK 293 cells were transfected with pcDNA3.1-eEF1A2(His)<sup>6</sup> , and after 24 h from transfection, cell extracts were analyzed by Western blot using commercial mouse anti-eEF1A antibody (A), anti-eEF1A2 antibody (B), and rabbit anti-His antibody (C). Lanes: −eEF1A2, non-transfected HEK 293 cells; +eEF1A2, HEK 293 cells transfected with pcDNA3.1-eEF1A2(His)<sup>6</sup> .

#### **2.2. Both eEF1A1 and eEF1A2 immuno-interact after pull-down**

The possible interaction between eEF1A isoforms was analyzed by pull-down experiment. To this purpose, GST-eEF1A1 (kindly supplied by C. Sanges, Wurzburg, Germany [21]) and pcDNA3.1-eEF1A2(His)<sup>6</sup> constructs were co-transfected in HEK 293 cells and, after 24 h from transfection, cell extracts were analyzed by Western blot following GST-agarose and Ni-NTAagarose pull-down. As shown in **Figure 2**, compared to controls, GST pull-down of co-transfected cells showed the presence of a band of 54 kDa corresponding to the size of eEF1A2(His)<sup>6</sup> (**Figure 2A**, lane 2), whereas Ni-NTA pull-down showed the presence of a band of about 78 kDa corresponding to the size of the construct GST-eEF1A1 (**Figure 2B**, lane 2). **Figure 2B** (lane 3) also shows the presence of a band of about 26 kDa corresponding to the GST protein. This finding suggested that GST by itself somehow interacted with Ni-NTA matrix; thus, the result shown in line 2 could be partly due to an interaction of the GST moiety present in GST-eEF1A1 with Ni-NTA and not with eEF1A2. Therefore, to further confirm the interaction between eEF1A isoforms, a different approach was undertaken after transfection of HEK 293 cells with pcDNA3.1-eEF1A2(His)<sup>6</sup> . In fact, as reported in **Figure 2C**, compared to cells transfected with pcDNA3.1 empty vector, cells transfected with eEF1A2(His)<sup>6</sup> showed, after Ni-NTA pull-down of cell extracts, the presence of a band of 54 kDa that was recognized by the specific anti-eEF1A1 (prepared as already reported [22]) (**Figure 2C**-a, lane 2) and anti-eEF1A2 (**Figure 2C**-b, lane 2) antibodies, the latter confirmed also with anti-His antibody (**Figure 2C**-c, lane 2).

The intracellular colocalization of eEF1A1 and eEF1A2 was first analyzed by confocal microscopy. As shown in **Figure 3**, HEK 293 cells after 48 h from transfection with pcDNA3.1-

panels (merged image, **Figure 3D**) showed that both eEF1A isoforms exhibited a cytoplasmic

gated by sensitized emission FRET method. FRET effect was performed by confocal microscope

colocalization with specific signals more intense at the level of the plasma membrane.

**2.4. FRET analysis showed that both eEF1A1 and eEF1A2 interact in HEK 293 cells**

The interaction between endogenous eEF1A and transfected eEF1A2(His)6

construct revealed that both endogenous eEF1A (**Figure 3A**) and transfected

Cellular Interaction of Human Eukaryotic Elongation Factor 1A Isoforms

http://dx.doi.org/10.5772/intechopen.74733

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(**Figure 3B**) shared a cytoplasmic localization. The superimposition of the two

was further investi-

in HEK 293 cells. HEK 293 cells were transfected with pcDNA3.1-

, and after 48 h from transfection, cells were analyzed by confocal microscopy. (A) eEF1A1, (B) eEF1A2, (C)

**2.3. Both eEF1A1 and eEF1A2 colocalize in HEK 293 cells**

**Figure 3.** Colocalization of eEF1A1 and eEF1A2(His)<sup>6</sup>

nuclear staining, and (D) merged images.

eEF1A2(His)6

eEF1A2(His)6

eEF1A2(His)6

**Figure 2.** Co-transfection of GST-eEF1A1 and eEF1A2-His in HEK 293 cells. GST-eEF1A1 and pcDNA3.1-eEF1A2(His)<sup>6</sup> were cotransfected in HEK 293 7 cells. After 24 h, the cells were harvested, lysed, and analyzed after GST pull-down with antibody anti-His (A) and after Ni-NTA pull-down with anti-GST antibody (B). (A) Lanes: 1, non-transfected cells; 2, cells transfected with GST-eEF1A1 and pcDNA3.1-eEF1A2(His)<sup>6</sup> ; 3, cells transfected with GST and pcDNA3.1 eEF1A2(His)6 ; 4, GST-agarose alone. (B) Lanes: 1, non-transfected cells; 2, cells transfected with GST-eEF1A1 and pcDNA3.1-eEF1A2(His)<sup>6</sup> ; 3, cells transfected with GST and pcDNA3.1-eEF1A2(His)<sup>6</sup> ; 4, Ni-NTA alone. (C) pcDNA3.1 eEF1A2(His)6 was co-transfected in HEK 293 7 cells. After 24 h, the cells were harvested, lysed, and analyzed after Ni-NTA pull-down with anti-eEF1A1, anti-eEF1A2, and anti-His antibody. (a–c) Lanes: 1, cells transfected with empty vector; 2, cells transfected with pcDNA3.1-eEF1A2(His)<sup>6</sup> .

pull-down of cell extracts, the presence of a band of 54 kDa that was recognized by the specific anti-eEF1A1 (prepared as already reported [22]) (**Figure 2C**-a, lane 2) and anti-eEF1A2 (**Figure 2C**-b, lane 2) antibodies, the latter confirmed also with anti-His antibody (**Figure 2C**-c, lane 2).
