Proteomic amalysis in Angiotensin signaling

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Intracellular signaling constitutes a complicated network, which are regulated by specific protein-protein interactions. Therefore identification of protein complexes is the key to understanding cellular functions. The crosstalk of Angiotensin signaling is mediated, in part, through the activation of Akt. The Akt-driven signaling network is capable of regulating cellular processes by modulating transcription and translation, and by modifying proteins at the posttranslational level. To elucidate the crosstalk of Angiotensin signaling, we searched for the binding molecule of Akt1. Here, we made attempts to perform affinity purification and proteomic analysis to elucidate the role of Akt in AngiotensinII signaling.We explored the use of strep-tag method for the isolation of Akt1 complexes from rAECs. The strep-Akt1 introduced into the rAECs by adenoviral vectors, and the cells were stimulated by AnguitensinII, Angiotensin receptor blocker, and ACE inhibitor for 10-30 min. Lysates expressing the strep-tagged Akt1 were then passed through a strep-Tactin column and bound proteins were specifically eluted using a desthiobiotin. The purified protein assemblies were separated by denaturing gel electrophoresis, and individual bands were digested by trypsin, analyzed by matrix-assisted laser desorption/vionization-time-of-flight mass spectrometry (MALDI-TOF MS) and identified by database search algorithms. As a result, at least 50 kinds of interaction partners were systematically purified and identified. Some of these proteins were changed the expresion level depend on stimulation. These included enzymes, protein kinases, translational regulators, cytoskeletal proteins, and hypothetical proteins. These results suggest that Angiotensin signaling has effects on the many biomolecular response and physiological consequence of vascular endothelium functions. This methodology would provide a new tool for chemical proteomics in medicinal science.