Investigating 14-3-3 Protein Subcellular Localization, Colocalization with Subcellular Markers and Interaction with Rac1

14-3-3 proteins are a group of widely expressed, highly conserved homo/heterodimeric acidic proteins. They usually bind to serine/threonine phosphorylated proteins, but also bind to proteins in a phosphorylation-independent manner. 14-3-3 proteins have over 200 binding partners. 14-3-3 proteins are involved in multiple cellular processes acting mainly as a scaffold protein. Mammals have seven 14-3-3 isoforms (α/β, ε, η, γ, σ, τ/, and δ/ζ), which are encoded by separate genes and are expressed on different chromosomes. While the existence of multiple isoforms may represent one more level of regulation in 14-3-3 signaling, knowledge regarding the isoform-specific functions of 14-3-3 proteins is very limited. Determination of the subcellular localization of the different 14-3-3 isoforms could give important clues into their specific functions. Most of the subcellular localization studies have been done in yeast, flies and plants and little isoform-specific subcellular localization studies have been done in mammals. By using immunocytochemistry, subcellular fractionation, and immunoblotting, I studied the subcellular localization of the total 14-3-3 protein and each of the seven 14-3-3 isoforms, their redistribution throughout the cell cycle and their translocation in response to EGF. In this thesis, I showed that 14-3-3 proteins are broadly distributed throughout the cell and associated with many subcellular organelles/structures including the plasma membrane, endosomes, mitochondria, endoplasmic reticulum, nucleus, centrosomes, microtubules, and actin fibers. I conclude that different isoforms of 14-3-3 have distinct subcellular localizations, which suggest their distinct cellular functions. I then focused my research to identify the novel binding partners of 14-3-3 proteins. Rac1, a member of the Rho GTPases, promotes the reorganization of actin filament polymerization in lamellipodia and membrane ruffles. It is interesting to notice that both 14-3-3 proteins and Rho GTPases regulate cytoskeleton remodeling and cell migration, which suggests a possible interaction between the signaling pathways. Indeed, previous research has only shown indirect interactions between 14-3-3 proteins and various Rac1 regulators and effectors. However, it is not clear if 14-3-3 proteins bind to Rac1 directly. Using co-immunoprecipitation, I show in this thesis that 14-3-3 proteins bind to Rac1 through serine 71 in a phosphorylation-dependent manner.