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. 2015 Jun 5;290(29):17879–17893. doi: 10.1074/jbc.M115.640821

FIGURE 6.

FIGURE 6.

Pterosin B represses SIK3 signaling via the C-terminal regulatory region. A, HEK293 cells were transformed with the MEF2 or CRTC2 reporter together with the SIK1–3 expression vectors and treated with pterosin B (300 μm) for 36 h. The fold differences in the reporter activities by the pterosin B treatment are indicated (n = 3, means and S.D.). B, in vitro kinase assay. The GST-SIK3 enzyme was expressed in HEK293 cells, purified with a glutathione resin, and incubated with compounds, the coumarin-labeled CRTC2 peptide, and 1 mm ATP for 1 h. The phosphorylated and nonphosphorylated peptides were separated by electrophoresis. C, HEK293 cells overexpressing GST-SIK3 (WT and T163A mutant) were treated with pterosin B (300 μm) for 36 h, and then the GST-SIK3 were purified and subjected to the in vitro kinase assay and Western blot analysis. D, the upper diagram shows phosphorylation sites in SIK3. HEK293 cells were transformed with reporters together with the SIK3 expression vectors as A. E, the same experiments with SIK3-phoshorylation site mutants were performed.