The regulation of phospholipid signaling and actin cytoskeletal remodeling by the eukaryotic translation elongation factor eEF1A2

Abstract

The regulation of the actin cytoskeleton is vital in several cellular and physiological processes from cell movement and phagocytosis to embryonic development and immune response. Thus, it is imperative to not only understand the mechanisms that regulate actin cytoskeletal dynamics but to also identify novel signaling networks and proteins that impinge upon these pathways. Here, we identify the eukaryotic elongation factor, eEF1A2, as a mediator of actin cytoskeleton remodeling through the phosphatidylinositol signaling pathway. EEF1A2, the gene encoding eukaryotic elongation factor 1 alpha 2 (eEF1A2), is one of two functional transcripts of EEF1A . During protein translation, eEF1A2 binds to amino-acylated tRNAs and recruits them to the ribosome. Aside from protein synthesis, eEF1A proteins from several genera and species associate with the actin cytoskeleton by binding and bundling actin. This suggests that eEF1A proteins may regulate actin cytoskeleton dynamics in addition to, or independent of, protein translation. Furthermore, recent work has identified EEF1A2 as an oncogene of the ovary, lung and breast, however the mechanism by which eEF1A2 contributes to oncogenesis has yet to be elucidated. The work presented in this thesis provides a mechanism by which eEF1A2 promotes oncogenesis. We first demonstrate that eEF1A2 can regulate phospholipid signaling by interacting with, and activating, phosphatidylinositol-4 kinase III beta (PI4KIIIbeta), a lipid kinase that is important in the phosphatidylinositol signaling pathway. Through a series of biochemical, molecular, and imaging assays, we describe and detail this interaction and provide evidence that eEF1A2-mediated PI4KIIIbeta activation results in an increased pool of phosphatidylinositol-4 phosphate (PI4P), the product of PI4KIIIbeta lipid kinase activity. We then go on show that the interaction between eEF1A2 and PI4KIIIbeta leads to the production of finger-like, actin-based membrane protrusions termed filopodia. Specifically, eEF1A2-mediated filopodia formation occurs in a phosphatidylinositol-4,5 bisphosphate (PI(4,5)P2)- and Cdc42-dependent manner. Furthermore, we show that PI4KIIIbeta activation is necessary for eEF1A2-mediated filopodia development. Lastly, we describe some of the ways eEF1A2 regulates breast oncogenesis. We show that ectopic eEF1A2 expression in breast cancer cells increases in vitro growth rates, inhibits anoikis, and activates the Akt serine-threonine kinase. Importantly, eEFIA2 is shown to be a valid therapeutic target as ablation of eEF1A2 decreases tumour formation in mice. Taken together, the work presented in this thesis provides new insights into ways eEF1A2 contributes to oncogenesis and provides a mechanism as to how eEF1A2 regulates the actin cytoskeleton.