In‐cell Cross‐linking and Identification of the Nuclear Actor of Activated T‐cells 5 Protein Interactome Using the Mass Spectrometry‐cleavable Covalent Cross‐linker DC4 (1182.7)

The Nuclear Factor of Activated T‐cells 5 (NFAT5) transcription factor undergoes hypertonicity‐induced nuclear localization and regulation of the transcription of osmoprotective genes. Its structure, function, compartmentalization, DNA‐, and protein‐protein interactions are influenced by the various cellular proteins that interact with it, including a number of kinases and phosphatases. Prior in vitro NFAT5 studies and ongoing in vitro peptide affinity chromatography indicate that NFAT5 is involved in a large number of protein‐protein interactions. To validate these in vitro results and to ascertain which interactions are physiologically relevant, we performed in‐cell protein crosslinking experiments. We used the collision induced dissociation (CID)‐cleavable cross linker DC4 in NFAT5‐transfected HEK 293 cells. Protein crosslinking was followed by column and/or gel purification of NFAT5 complexes, protease digestion, LC‐MS‐MS and protein identification analyses. The proteins identified by in‐cell analysis were cross‐referenced with the results of our in vitro experiments to help gain domain‐specific information on NFAT5 protein interactions.Grant Funding Source: Supported by the Intramural Research Program of the NIH, National Heart, Lung, and Blood Institute