**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 was further investigated by sensitized emission FRET method. FRET effect was performed by confocal microscope

**Figure 3.** Colocalization of eEF1A1 and eEF1A2(His)<sup>6</sup> in HEK 293 cells. HEK 293 cells were transfected with pcDNA3.1 eEF1A2(His)6 , and after 48 h from transfection, cells were analyzed by confocal microscopy. (A) eEF1A1, (B) eEF1A2, (C) nuclear staining, and (D) merged images.

pcDNA3.1-eEF1A2(His)<sup>6</sup>

of eEF1A isoforms.

**4. Conclusions**

**5. Materials and methods**

**5.1. Cell culture and transfection**

were grown at 37°C in a 5% CO<sup>2</sup>

cillin, 100 mg/ml streptomycin, and 1% l-glutamine.

, and the interaction of the recombinant eEF1A2(His)6

Cellular Interaction of Human Eukaryotic Elongation Factor 1A Isoforms

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

enous eEF1A1 was assessed by pull-down, confocal microscopy and FRET analysis. The results obtained showed that the endogenous eEF1A1 and the expressed eEF1A2 interacted in HEK 293 cells at the level of both cytoplasm and plasma membrane. Moreover, the FRET image highlighted a more intense signal at the level of the plasma membrane. These data confirmed those reported in our previous work [22], thus strongly confirming the association in the cells

The homodimer association of eEF1As has recently emerged from the crystallization of rabbit eEF1A2 [27] or as proposed in *Tetrahymena*, in order to explain actin bundling essential for the regulation of actin cytoskeleton and cell morphology during several cellular processes [16]. The possible association between eEF1A isoforms was instead proposed by Sanges et al. [21] in studying the control of eEF1A function in cancer cells *via* phosphorylation and by Lee et al. [28] in studying the interaction of eEF1A2 with the tumor suppressor protein p16INK4a. Since eEF1A1 and eEF1A2 display a very high amino acid sequence identity (above 97%), the overall structures appear quite similar, as can be predicted by bioinformatic analysis at the GRAMM-X docking Web Server v.1.2.0 [29, 30], using rabbit eEF1A2 (PDB 4C0S chain A) as template [27]. These considerations suggest that both eEF1A1 and eEF1A2 complexes are present in the cells either as homodimer or as heterodimer. These complexes are most likely

Because eEF1A dimers are involved in actin bundling [31, 32], it emerges that the fraction of eEF1A as dimer is mostly involved in the actin cytoskeleton rearrangement. Therefore, the cellular distribution of eEF1A molecules between monomeric and dimeric form regulates the functional role of eEF1A in translation or in actin bundling. Because actin chains and translational system coexist in the cells and maybe also functionally dependent [33, 34], the transition "monomer-dimer-monomer" of eEF1A should be relatively easy depending on the cell conditions [35]. This interconversion may be regulated by the reversible posttranslational modifications of eEF1A [36] and its interactions with the protein partners such as Raf kinases [20, 21]. Therefore, it is possible that in cells coexpressing both isoforms, like cancer cells, eEF1A heterodimer formation could also be important for cytoskeleton rearrangements rather than for some phos-

phorylation catalysis most likely occurring during cell survival and apoptosis [20, 21].

HEK 293 cells, obtained from the American Type Tissue Collection (Rockville, MD, USA),

(Gibco, Monza, Italy) supplemented with 10% heat-inactivated FBS (GIBCO), 100 U/ml peni-

atmosphere in Dulbecco's modified Eagle medium (DMEM)

associated with regulatory noncanonical functions of eEF1As.

with endog-

73

**Figure 4.** FRET analysis of the interaction between eEF1A1 and eEF1A2. Representative pseudocolor images of cells labeled with rabbit anti-eEF1A1 and mouse anti-His primary antibodies followed by FITC and TRITC secondary-labeled antibodies.

that allowed discriminate proteins that colocalize in the same cellular compartment from those that are instead involved in specific molecular interactions. FRET effects were calculated using ImageJ plug-in software [24]. **Figure 4** shows the representation of the FRET effects where the blue color is indicated at low signal, whereas yellow-white color designated a high signal. The images clearly showed the interaction between eEF1A1 and transfected eEF1A2(His)6 within the cytoplasm with specific signals more intense especially at the level of the plasma membrane.
