Abstract
Tyrosine autophosphorylation plays a crucial regulatory role in the kinase activities of fibroblast growth factor receptors (FGFRs), and in the recruitment and activation of downstream intracellular signaling pathways. Biophysical and biochemical investigations of FGFR kinase domains in membrane environments offer key insights into phosphorylation mechanisms. Hence, we constructed nickel chelating nanodiscs based on a 22-residue peptide. The spontaneous anchoring of N-terminal His6-tagged FGFR1c kinase domain (FGFR1K) onto peptide nanodiscs grants FGFR1K orientations occurring on native plasma membranes. Following membrane incorporation, the autophosphorylation of FGFR1K, as exemplified by Y653 and Y654 in the A-loop and the total tyrosine phosphorylation, increase significantly. This in vitro reconstitution system may be applicable to studies of other membrane associated phenomena.