Besides its canonical role in protein synthesis, the eukaryotic translation elongation factor 1A (eEF1A) is also involved in many other cellular processes such as cell survival and apoptosis. We showed that eEF1A phosphorylation by C-Raf in vitro occurred only in the presence of eEF1A1 and eEF1A2, thus suggesting that both isoforms interacted in cancer cells (heterodimer formation). This hypothesis was recently investigated in COS-7 cells where fluorescent recombinant eEF1A isoforms colocalized at the level of cytoplasm with a FRET signal more intense at plasma membrane level. Here, we addressed our attention in highlighting and confirming this interaction in a different cell line, HEK 293, normally expressing eEF1A1 but lacking the eEF1A2 isoform. To this end, His-tagged eEF1A2 was expressed in HEK 293 cells and found to colocalize with endogenous eEF1A1 in the cytoplasm, also at the level of cellular membranes. Moreover, FRET analysis showed, in this case, the appearance of a stronger signal mainly at the level of the plasma membrane. These results confirmed what was previously observed in COS-7 cells and strongly reinforced the interaction among eEF1A isoforms. Moreover, the formation of eEF1A heterodimer in cancer cells could also be important for cytoskeleton rearrangements rather than for phosphorylation, most likely occurring during cell survival and apoptosis.
Part of the book: Protein-Protein Interaction Assays