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Fig. 2
Control of Src kinase activity with uniRapR domain. (A) Schematic representation of activity control with the uniRapR domain. (B) Root mean square fluctuations of the ATP binding site (gray structure) based on multiple equilibrium DMD simulations for WT Src (black), apo (red), and holo (green) uniRapR-inserted Src (P < 0.01). (C) HEK293T cells expressing the Src-uniRapR-cerulean-myc construct were treated with different concentrations of rapamycin (0–2 µM), and lysates were assayed for expression of the construct with Western blotting by using anti-GFP. The construct was pulled down with anti-myc and mixed with the paxillin substrate in the presence of ATP for 10 min. Reaction suspensions were blotted and probed with anti-myc and anti–pY31-paxillin to confirm binding and phosphorylation of the substrate, respectively. (D) As controls, constitutively active Src (YF) without the uniRapR domain, kinase dead (YF/KD), Y271A and L1polyP Src mutants with the uniRapR domain, and our previous dimerization-based switch were tested. (E) Y271A and L1polyP substitutions shown on the Src-uniRapR model.