For directly imaging the bindings between chemical drugs and their target protein kinases in live cells using a redistribution assay, we have developed a methodology. Briefly, the HaloTag protein (HT) (or Streptavidin (SA)) was fused to Protein kinase C (PKC), which enables the chemical drug and its target protein kinase to cotranslocate from the cytoplasm to the plasma membrane. PKC is well known to translocate from the cytoplasm to the plasma membrane in response to physiological stimuli, as well as exogenous ligands such as phorbol esters. The HT is a mutant of a hydrolase protein that efficiently forms a covalent bond with the HT ligands such as aliphatic halogenated compounds. In particular, we initially modified the chemical drugs (e.g. Dasatinib) with 6-chlorohexyloxyethane. The resulting compounds were cell-membrane permeable, and binding both HT and its target protein kinases. To verify our approach, the fusion construct PKC-mRFP-HT and each of the eGFP-tagged dasatinib target kinases (CSK, SRC, and LYN) were transiently cotransfected into HEK-293T cells and the cells were treated with Halotag Ligand-labelled dasatinib. When the exogenous ligand phorbol 12-myristate 13-acetate (PMA) was added, both PKC fusion and eGFP-tagged dasatinib target kinase were cotranslocated to the plasma membrane. To image the FKBP12-rapamycin-FRB complex, PKC-mRFP-SA/eGFP-FRB/TaqBFP-FKBP12 cotransfected cells were pretreated with biotin-labelled rapamycin before PMA treatment. As expected, treatment with rapamycin induced an interaction between FKBP12 and FRB, FKBP12 and FRB were cotranslocated to the plasma membrane.

Imaging of bindings between chemical drug and its target protein kinases by redistribution assay in live cells

248th National Meeting (2014)

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