Nuclear translocation of nfat family proteins in human reporter cell lines

Official URL: http://risc01.sabanciuniv.edu/record=b1301365 (Table of Contents)

Nuclear factor of activated T-cells (NFAT) proteins are transcription factors evolutionary related to the NFκB/Rel family. The NFAT family consists of five proteins and several isoforms that regulate the expression of various immune system genes. The mRNAs that encode NFAT proteins, which were first thought to be T-cell-specific, vary in distribution between different cells and tissues. In dormant cells, NFAT proteins are highly phosphorylated and reside in the cytoplasm. Upon phosphorylation by a signal activated calcineurin phosphatase, NFATs are imported to the nucleus. NFAT proteins are exported back out of the nucleus by NFAT kinases such as CK1 and GSK3. This study aimed to investigate the domains necessary for the subcellular translocation of NFAT transcription factor proteins upon PMA/ionomycin treatment. The calcium phosphate transient transfection method was used to express different NFAT family members in mammalian cells. We found that ionomycin treatment results in the nuclear translocation of NFAT2 while NFAT3 does not follow this rule. We generated reporter human embryonic kidney 293 cell lines that express green fluorescent protein (GFP) upon NFAT nuclear translocation. Fluorescent microscopy and flow cytometry experiments were performed and showed that the nuclear localization of NFAT2 begins within 15 minutes after PMA/ionomycin stimulation, with most of the proteins entering the nucleus after two hours. We identified a domain in the NFAT2 protein called the regulatory domain which was important for translocation. Protein-protein interactions likely play a crucial role in NFAT nuclear import. Future experiments using biochemical techniques may help to bring these proteins to light.