Light regulation of protein dimerization and kinase activity in living cells using photocaged rapamycin and engineered FKBP

Abstract

We developed a new system for light-induced protein dimerization in living cells using a photocaged analogue of rapamycin together with an engineered rapamycin binding domain. Using focal adhesion kinase as a target, we demonstrated successful light-mediated regulation of protein interaction and localization in living cells. Modification of this approach enabled light-triggered activation of a protein kinase and initiation of kinase-induced phenotypic changes in vivo.

Figure 1

Photocaging of rapamycin. (a) Structure of rapamycin (Rap) and its synthetic transformation into caged rapamycin (pRap) through selective acylation of the C-40 hydroxyl group with nitro-piperonyloxycarbonyl N-hydroxysuccinimide carbonate (NPOC-NHS). (b) Schematic of the light-induced heterodimerization of the proteins FRB and iFKBP using pRap. (c) Crystal structure of the ternary complex between rapamycin, FKBP12 (green), and FRB (blue). The 2.65 Å hydrogen bond (possibly mediated through a water molecule) between Gln53 of FKBP12 and the C-40 hydroxyl group of rapamycin is indicated. PDB 2FAP.

Figure 2

Light-regulated dimerization of iFKBP and FRB. (a) Positions of iFKBP insertions into FAK. (b–d) HEK293T cells co-transfected with GFP-FRB and either myc-iFKBP-FAK (b, c) or myc-FAK-iFKBP413 (d) were treated with either rapamycin (0.5 µM) or the indicated concentrations of pRap. Ten minutes after addition of pRap or Rap, cells were irradiated with 365 nm UV light for 1 min (b, d) or 5 min (c), and incubated for 1 hour. Control cells were not irradiated. Myc-iFKBP-FAK was immunoprecipitated from cell lysates using anti-myc antibody and co-immunoprecipitation of GFP-FRB was detected by Western Blot using anti-GFP antibody. (e) HeLa cells co-transfected with GFP-FAK-iFKBP413 and mCherry-FRB were treated with pRap (20 µM) for 30 min, followed by UV irradiation (365 nm, 2 min). TIRF images were taken before and after irradiation. (f) HeLa cells co-transfected with GFP-RapR-FAK and mCherry-FRB were treated with pRap (5 µM) for 30 min, followed by UV irradiation (365 nm, 2 min). TIRF images were taken before and after irradiation.

Figure 3

Light-mediated activation of a protein kinase. (a) Schematic of RapR-FAK regulation by pRap. (b) Myc-RapR-FAK kinase was co-expressed with GFP-FRB in HEK293T cells. Cells were treated with the indicated amount of rapamycin (Rap), caged rapamycin (pRap), or DMSO (control). The indicated samples were exposed to UV light (365 nm, 1 min). All cells were incubated at 37 °C for 1 hour after treatment. Myc-RapR-FAK was immunoprecipitated using an anti-myc antibody and tested in a kinase assay using an N-terminal fragment of paxillin as a substrate. The level of phosphorylation of paxillin on Tyr31 (probed with anti-phospho-Tyr31 paxillin antibody) indicates the kinase activity. (c, d) HeLa cells co-transfected with GFP-RapR-FAK and Cherry-FRB were treated with pRap (1 µM) and imaged before (c) and after (d) UV irradiation (365 nm, 1 min). Arrows indicate formation of large dorsal ruffles stimulated by activated RapR-FAK.

Figure 4

Molecular dynamics simulations of the FKBP12-pRap-iFKBP interaction. (a) Tube representations of FKBP12 (left) and iFKBP (right) with pRap in the binding pocket. Warmer colors and thicker backbones correspond to fluctuations within the structure. Note the greater mobility of iFKBP. (b) Binding interfaces of the pRap, FRB, and FKBP12 or iFKBP complexes. For iFKBP (right) the interface area is reduced due to loss of contacts between the Asp41-Leu49 segment and FRB. In the iFKBP complex, residues that were in contact for the FKBP12 complex are marked in light brown. The segment deleted from FKBP12 to produce iFKBP is marked with dark color in FKBP12.