Aim of this project is to develop a method for capturing kinase-substrate pairs in living mammalian cells using unnatural amino acid mutagenesis and affinity crosslinking. Photocrosslinking amino acids (containing benzophenone or azide groups) will be introduced site-specifically into the backbone of target proteins using ad hoc developed orthogonal tRNA/synthetase pairs functional in mammalian cells. By introducing the photocrosslinker at suitable positions into the substrate binding region of a protein kinase, we expect to crosslink its natural substrates in response to cell stimulation and UV irradiation. Likewise, by introducing a photocrosslinker close to the phosphorylation site of a kinase substrate we will aim at capturing it upstream kinase. Once proofed the feasibility of the approach using the PKA/CREB system, we will apply such strategy to identify unknown kinases-substrate pairs involved in signaling pathways regulating fundamental cellular processes (BRCA1 and NF-kappaB pathway). This will represent a new general method for capturing transient protein-protein interactions in vivo by applying a minimal perturbation to the natural system, which will respond to a need of the scientific community, since methods for studying protein-protein interactions available nowadays mostly involve non-native conditions. The use of orthogonal tRNA/synthetase pairs for unnatural amino acid mutagenesis is a very recently developed technique, which is applied in very few laboratories in the world, and which has a tremendous potential for enabling the study of protein structure and function directly in the living cell. In the outgoing phase the fellow intends to learn this technique and explore new possibilities of its application. This knowledge will be applied in the return phase to study a major research target of the return host. The establishment of this technique in European countries will increase the competitiveness of the European research in the biomedical field.
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