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Fig. 2.
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Fig. 3.
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The effect of Sufu on Gli3 ovalbumin 324-338 by GSK3β was also examined with an in vitro kinase assay in which PKA-primed Gli3 was used as a substrate ( Fig. 2B). Whereas GSK3β alone induced only a slight phosphorylation of GST-Gli3(18–102 cool , the phosphorylation level was significantly augmented in the presence of Sufu. Sufu alone showed no significant effect on the phosphorylation level. Furthermore, Sufu did not increase the autophosphorylation level of GSK3β, suggesting that Sufu did not affect the kinase activity of GSK3β.
It has been reported that the processing of three Gli3 mutants, in which each GSK3β-site Ser residue (G2, G3, and G4 in Fig. 2C) was substituted with Ala, is impaired, indicating the critical importance of each Ser in Gli3 processing [10]. To examine whether Sufu promotes phosphorylation of these GSK3β sites, three GST-Gli3(18–102 cool mutants were prepared in which each Ser was substituted with Ala, and an in vitro kinase assay was performed ( Fig. 2D). Sufu induced a 2.4-fold increase in the phosphorylation level of wild type Gli3. However, the stimulatory effect of Sufu was almost abolished in the S903A mutant (1.2-fold increase), and was significantly reduced in the S861A mutant (1.6-fold increase). Slight decreases in the effect of Sufu were also observed in the S873A mutant (2.1-fold). These results suggest that Sufu mainly promotes phosphorylation of Ser903, which is required for Gli3 processing.