Figure 3
Inhibitory effects of orobol on CK1ε kinase activity. (A) Orobol was tested for CK1ε inhibitory activity in ten concentrations with 2-fold serial dilutions starting at 20 μM. (B) Orobol binds to CK1ε directly in vitro. The orobol binding was evaluated by immunoblotting using an antibody against CK1ε: lane 1, CK1ε; lane 2, CK1ε kinase bound to orobol-Sepharose 4B beads and lane 3, CK1ε precipitated with Sepharose 4B. (C) Orobol directly interacts with CK1ε in 3T3-L1 cell lysates. The CK1ε kinase bound to orobol was evaluated by immunoblotting: lane 1, CK1ε kinase in whole lysates of 3T3-L1 cells; lane 2, CK1ε kinase in lysates precipitated with Sepharose 4B beads; and lane 3, CK1ε in whole lysates of 3T3-L1 cells precipitated by orobol-Sepharose 4B beads. (D) Orobol binds to CK1ε in an ATP-competitive manner. CK1ε (0.2 µg) was incubated with ATP at the indicated concentrations (0, 10, or 100 μM) together with 100 µl orobol-Sepharose 4B beads or Sepharose 4B beads (negative control) added in reaction buffer to a final volume of 500 µl. The immunoprecipitated proteins were detected by immunoblotting with an antibody against CK1ε. Lane 2, negative control, showing that CK1ε does not bind to Sepharose 4B beads alone; lane 3: positive control, showing that CK1ε binds with orobol-Sepharose 4B beads. Presented signals from were cropped from one continuous Western blot which is displayed as Suppl. Figure (E,F) Model structure of CK1ε in complex with orobol (E) and the detailed interaction of the complex (F). Orobol (atomic color) binds to the ATP-binding site of CK1ε; PF4800567 (blue) is overlaid for comparison. The residues involved in the interaction with orobol are labeled and the hydrogen bonds are depicted as dotted lines.
